Compositions and soft tissue replacement methods

ABSTRACT

The invention relates to compositions comprising a hydrogel material that comprises a glycosaminoglycan polymer, adipose tissue and a compound of the structure 
                         
The composition is administered to a site of a soft tissue condition including those of breast tissue. The hydrogel material can be porous or of solid particles. The glycosaminoglycan polymer can be a hyaluronan polymer.

CROSS REFERENCE

This application claims priority pursuant to 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/444,078, filed Feb. 17, 2011, andis a continuation-in-part pursuant to 35 U.S.C. §120 to U.S.Non-Provisional patent application Ser. No. 13,193,744, now U.S. Pat.No. 8,697,056 filed on Jul. 29, 2011, which claims priority to U.S.Provisional Patent Application 61/375,144, filed on Aug. 19, 2010, eachof which is hereby incorporated by reference in its entirety.

BACKGROUND

Soft tissue replacement methods are commonly used for a wide variety ofclinical and cosmetic purposes. One use involves reconstructiveapplications that rebuild and restore a body part or structure tocorrect deformities from congenital anomalies, trauma, cancer,infections, disease, or medication side effects. The replacement tissueserves to support surrounding tissue and to maintain the normalappearance of the body. The restoration of this normal appearance has anextremely beneficial psychological effect on post-operative patients,alleviating much of the shock and depression that often followsextensive surgical procedures. Another use involves augmentationapplications that alter a body part or structure usually to improve itscosmetic or aesthetic appearance. Augmentation of the appearance alsohas beneficial psychological effects that improve self-esteem,well-being, and confidence of an individual. A third use involvesstructural applications that provide support to a body part or structureto improve its function, thereby alleviating a symptom associated with adisorder involving soft tissue loss. Examples of such disorders include,without limitation, stress urinary incontinence, fecal incontinence,vocal cord paralysis, vocal atrophy, vocal implantation, intubationtrauma, post-hemilaryngectomy defects, irradiation damage, lumbar discrepair, and plantar footpad repair.

Soft tissue replacement methods currently rely on two generalapproaches: 1) implantation of artificial or alloplastic fillers likesoft tissue implants, and injectable polymers and hydrogels; and 2)transplantation of tissue like tissue flaps and autologous tissuetransfers. A drawback to the use of artificial or alloplastic fillers itthat these inorganic materials lack any metabolic activity, do notbecome physiologically incorporated into the body, and as such,surrounding tissue and blood supply does not develop within theimplanted material. In addition, artificial or alloplastic fillers riskmigration and/or extrusion from the implant site. Furthermore, many ofthese fillers produce only temporary effects because the body rapidlyreabsorbs them. Fillers providing long-term effects frequently induce aforeign body response resulting in formation of an avascular, fibrouscapsule around the filler, which limits performance, distorts theaesthetic appearance of the surrounding area, and can cause pain to theindividual. Furthermore, long-term fillers, like soft tissue implants,do not remodel with the aging tissue resulting in a material that maynot be aesthetically acceptable as the patient's tissues undergo thenormal physiologic changes associated with aging.

Although the use of transplanted tissue avoids the problems associatedwith artificial or alloplastic fillers, drawbacks are also associatedwith these procedures. In these procedures, loss of transplanted tissuevolume over time as a result of its resorption by the body is a majorproblem. For example, transplantation of adipose tissue generallyresults in a loss of 20% to 90% of its volume within the first year.This tissue loss is unpredictable and is a result of poor survivaland/or regeneration from progenitor cells in the transplanted tissue dueto necrosis and a lack of vascular formation. With respect to adiposetissue, tissue breakdown is associated with traumatic rupture of thecells, avascular necrosis, apoptosis of the adipocytes, inflammationsecondary to apoptosis, fibrosis and contraction of the graft, and/ordelipidation of the adipocytes with subsequent volume loss. Failedtissue grafts sometime produce stellate and irregular nodules withcalcifications. As such, transplanted tissue methods are usuallyperformed two or three times to obtain the desired effect, resulting inmassive time and cost.

One of the major underlying causes for tissue breakdown seen intransplanted tissue methods is the lack of a blood supply sufficient tosupport the transplanted tissue. For example, alleviation of tissueischemia is critically dependent upon formation of new blood vessels.The growth of new blood vessels and associated vasculature or repair orremodeling of existing blood vessels and associated vasculature providescollateral circulation in and around an ischemic area, improves bloodflow, and alleviates the symptoms caused by the ischemia. Thus,compositions and methods that promote new blood vessel formation withina transplanted tissue are needed as this will improve the survival rateof such tissues, thereby achieving reliable long-term survival ofgrafted tissues.

The present specification provides novel compositions and soft tissuereplacement methods using compositions that reduce tissue volume loss byincreasing the survival rate of the transplanted tissue. This improvedsurvival rate is achieved by administering a compound that promotes newblood vessel formation, thereby ensuring that a blood supply adequate tosupport the transplanted tissue is established.

BRIEF SUMMARY

Thus, aspects of the present specification disclose a compositioncomprising adipose tissue, a hydrogel material, and a compound havingthe structure of formula I, a pharmaceutically acceptable salt thereof,or a pharmaceutically acceptable amine salt thereof,

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁶is CO₂H, CO₂R⁷, CON(R⁷)₂, CONHCH₂CH₂OH, CON(CH₂CH₂OH)₂, CH₂OR⁷,P(O)(OR⁷)₂, or

R⁷ is H or C₁₋₆ hydrocarbyl; X and Y are each independently selectedfrom H, OH, ═O, Cl, Br, I, or CF₃; Z¹ and Z² are each independentlyselected from CH or N; W¹ and W² are each independently selected fromCH, CH₂, optionally substituted aryl, or optionally substitutedheteroaryl; m is 0, 1, 2, 3, 4, 5, or 6; o is 0, 1, 2, 3, 4; p is 0 or1; and V is C₁₋₆ alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

Other aspects of the present specification disclose a compositioncomprising adipose tissue, a hydrogel material, and a compound havingthe structure of formula VI, a pharmaceutically acceptable salt thereof,or a pharmaceutically acceptable amine salt thereof

wherein each dashed line represents the presence or absence of a bond;Z² and Z³ are independently CH or N; Z¹, Z⁴, Z⁵, and Z⁶ are eachindependently CR⁹, CHR⁹, NH, O, or S; A is —(CH₂)₆—, or cis—CH₂CH═CH—(CH₂)₃—, wherein 1 or 2 carbons may be substituted with S orO; or A is —(CH₂)_(m)—Ar—(CH₂)_(o)— wherein Ar is optionally substitutedaryl or optionally substituted heteroaryl, the sum of m and o is from 1,2, 3, or 4, and wherein one CH₂ may be substituted with S or O; R⁸ is H,hydrocarbyl, or hydrocarbyl-OH; R⁹ is independently H, C₁₋₆ alkyl, OH,F, Cl, Br, I, or O; J is C₁₋₆ alkyl, C₁₋₆—O-alkyl, C═O, or CHOH; E isC₁₋₁₂ alkyl, R¹⁰, or —Y—R¹⁰ wherein Y is CH₂, S, or O, and R¹⁰ isoptionally substituted aryl or optionally substituted heteroaryl; and nis 0 or 1.

Other aspects of the present specification disclose a compositioncomprising adipose tissue, a hydrogel material, and a compound havingthe structure of formula IX

wherein X¹ and X⁵ are independently CH or N; X⁷ is NH, O, or S; X⁸ andX⁹ are independently CH or N; R¹³ is optionally substituted aryl oroptionally substituted heteroaryl; and R¹⁴ is C₁₋₆ alkyl, optionallysubstituted aryl, or optionally substituted heteroaryl.

Other aspects of the present specification disclose a compositioncomprising adipose tissue, a hydrogel material, and a compound havingthe structure of compound 24, a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable amine salt thereof.

Yet other aspects of the present specification disclose a method oftreating a soft tissue condition of an individual, the method comprisingthe step of administering a composition disclosed herein to a site ofthe soft tissue condition, wherein administration of the compositionpromotes formation of a blood supply sufficient to support thetransplanted tissue, thereby treating the soft tissue site. Non-limitingexamples of a soft tissue condition include breast imperfection, defect,disease and/or disorder, such as, e.g., a breast augmentation, a breastreconstruction micromastia, thoracic hypoplasia, Poland's syndrome,defects due to implant complications like capsular contraction and/orrupture; a facial imperfection, defect, disease or disorder, such as,e.g., a facial augmentation, a facial reconstruction, Parry-Rombergsyndrome, lupus erythematosus profundus, dermal divots, sunken cheeks,thin lips, nasal imperfections or defects, retro-orbital imperfectionsor defects, a facial fold, line and/or wrinkle like a glabellar line, anasolabial line, a perioral line, and/or a marionette line, and/or othercontour deformities or imperfections of the face; a neck imperfection,defect, disease or disorder; a skin imperfection, defect, disease and/ordisorder; other soft tissue imperfections, defects, diseases and/ordisorders, such as, e.g., an augmentation or a reconstruction of theupper arm, lower arm, hand, shoulder, back, torso including abdomen,buttocks, upper leg, lower leg including calves, foot including plantarfat pad, eye, genitals, or other body part, region or area, or a diseaseor disorder affecting these body parts, regions or areas; urinaryincontinence, fecal incontinence, other forms of incontinence; andgastroesophageal reflux disease (GERD).

Still other aspects of the present specification disclose a method oftreating a soft tissue condition of an individual, the method comprisingthe steps of a) administering the adipose tissue to a site of the softtissue condition; b) administering a composition comprising a compoundas disclosed herein to the site of the soft tissue condition; and c)administering a hydrogel material to the site of the soft tissuecondition, wherein administration of the compound promotes formation ofa blood supply sufficient to support the transplanted tissue, therebytreating the soft tissue site. Non-limiting examples of a soft tissuecondition include those described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 illustrate possible ways to prepare compounds disclosedherein.

DETAILED DESCRIPTION

Insufficient blood vessel formation in a repairing or otherwisemetabolically active tissue results in inadequate delivery of oxygen,nutrients, and other substances necessary to establish essentialphysiological functions to the area and promote wound healing. Theformation of blood vessels within a tissue may occur by angiogenesisand/or vasculogenesis. As used herein, the term “angiogenesis” refers toa physiological process involving the growth of new blood vessels frompre-existing vessels and includes sprouting angiogenesis, the formationof new blood vessel by sprouting off existing ones, and splittingangiogenesis (intussusception), the formation of new blood vessel bysplitting off existing ones. As used herein, the term “vasculogenesis”refers to a physiological process involving the de novo production ofnew blood-vessels by proliferating endothelial stem cells, and as such,the formation of new blood vessels when there were no pre-existing ones.

Blood vessel formation, whether angiogenesis or vasculogenesis, requiressignals from growth factors and other proteins that direct and controlthe process, such as, e.g., fibroblast growth factors (like FGF-1 andFGF-2), vascular endothelial growth factors (like VEGF-A and VEGF-C),angiopoietins (like Ang-1 and Ang-2), platelet derived growth factor(PDGF), monocyte chemotactic protein-1 (MCP-1) (also known as chemokine(C—C motif) ligand 2 (CCL-2)), transformation growth factor betas(TGF-β1, TGF-β2, TGF-β3, and TGF-β4), vascular cell adhesion molecules(like VCAM-1), matrix metalloproteinases (like MMP-2 and MPP-9),integrins, cadherins, plasminogen activators, plasminogen activatorinhibitors, and ephrin.

Of the factors listed above, two of the more critical factors for newblood vessel formation are FGF-2 and VEGF. One of the most importantfunctions of FGF-2 (or bFGF) is the promotion of endothelial cellproliferation and the physical organization of endothelial cells intotube-like structures, thus promoting angiogenesis and vasculogenesis.FGF-2 is a more potent factor in new blood vessel formation than VEGF orPDGF. As well as stimulating blood vessel growth, FGF-2 is an importantfactor in wound healing. This factor stimulates proliferation offibroblasts and endothelial cells that give rise to new vessel formationand developing granulation tissue; both increase blood supply and fillup a wound space/cavity early in the wound-healing process.

VEGF is another major contributor to new blood vessel formation. VEGFcauses a massive signaling cascade in endothelial cells resulting in therelease of many other factors known to be responsible for new bloodvessel formation. For example, binding to VEGF receptor-2 (VEGFR-2)starts a tyrosine kinase signaling cascade that stimulates theproduction of factors that variously stimulate vessel permeability(eNOS, producing NO), proliferation/survival (FGF-2), migration(ICAMs/VCAMs/MMPs) and finally differentiation into mature bloodvessels. In addition, in vitro studies demonstrated that uponstimulation by VEGF and FGF-2, endothelial cells would proliferate,migrate, and eventually form tube structures resembling capillaries.

The compounds disclosed herein, and compositions comprising suchcompounds, upregulate both FGF-2 and VEGF (see Example 2). In addition,both incisional and ulcer wound models demonstrate that the upregulationof FGF-2 and VEGF results in an improved wound healing response (seeExample 2).

Without wishing to be bound by any particular theory, the compounds,compositions and methods disclosed herein increase the survival rate oftransplanted tissue by promoting new blood vessel formation within thetransplanted tissue. Administration of a compound, either as acomposition with the transplanted tissue or as a prior or subsequentadministration to the transplantation procedure, upregulate factorsessential to new blood vessel formation, like FGF-2 and VEGF. Thisupregulation initiates the signaling cascade in endothelial cellsresponsible for the formation of new blood vessel. This initiationresults in the formation of new blood vessels that, in turn, provideoxygen, nutrients, and other substances necessary to establish essentialphysiological functions to the transplanted tissue. This adequate bloodsupply supports the establishment, growth and survival of thetransplanted tissue. Thus, the compounds, compositions and methodsdisclosed herein ameliorate or prevent one of the major underlyingcauses for the tissue breakdown of transplanted tissue, namely the lackof a blood supply sufficient to support the transplanted tissue. Inaddition, the compounds and compositions disclosed herein are superiorto the mere addition of factors critical to new blood vessel formation,like FGF-2 and VEGF, because these small molecules are more stable andless costly then these protein-based therapies. Furthermore, formationof new blood vessels can soften scar tissue, capsular contracture byfacilitating the remolding of fibrous tissue into normal soft tissuetypical of the affected region.

Aspects of the present specification provide, in part, a compositioncomprising adipose tissue. As used herein, the term “adipose tissue” issynonymous with “fat” or “fatty tissue” and refers to a loose fibrousconnective tissue comprising fat cells (adipocytes) and multiple typesof regenerative cells. Adipose tissue may comprise brown and/or whiteadipose tissue taken from any body site, such as, e.g., subcutaneous,omental/visceral, interscapular, or mediastinal. It may be obtained fromany organism having adipose tissue, or the adipose tissue used may befrom a primary cell culture or an immortalized cell line.

Adipose tissue may be collected from the same individual who isundergoing the soft tissue replacement procedure (autograft), from adonor individual who is not the same individual as the one undergoingthe soft tissue replacement procedure (allograft), or from an animalsource (xenograft). As used herein, the term “autotransplantation”refers to the transplantation of organs, tissues, or cells from one partof the body to another part in the same individual, i.e., the donor andrecipient are the same individual. Tissue transplanted by such“autologous” procedures is referred to as an autograft orautotransplant. As used herein, the term “allotransplantation” refers tothe transplantation of organs, tissues, or cells from a donor to arecipient, where the donor and recipient are different individuals, butof the same species. Tissue transplanted by such “allologous” proceduresis referred to as an allograft or allotransplant. As used herein, theterm “xenotransplantation” refers to the transplantation of organs,tissues, or cells from a donor to a recipient, where the donor is of adifferent species as the recipient. Tissue transplanted by such“xenologous” procedures is referred to as a xenoograft orxenotransplant.

Adipose tissue can be collected by any procedure that can harvestadipose tissue useful for the compositions and methods disclosed herein,including, without limitation a liposuction (lipoplasty) procedure or alipectomy procedure. Procedures useful for collecting adipose tissueshould minimize the trauma and manipulation associated with adiposetissue removed. Adipose tissue may be harvested from any suitableregion, including, without limitation, a mammary region, an abdominalregion, a thigh region, a flank region, a gluteal region, a trochanterregion, or a gonadal region. Procedures useful for collecting adiposetissue are well known to a person of ordinary skill in the art. Theselected procedures may be performed concomitantly with liposculpture.

A liposuction procedure harvests adipose tissue by aspirating the tissueusing a cannula. The cannula may be connected to a syringe for manualaspiration or to a power assisted suction device, like an aspirator,adapted to collect the adipose tissue into a vacuum bottle. Aliposuction procedure does not maintain an intact blood supply of theharvested tissue. The syringe may be a 10, 20 or 60 mL syringe fittedwith a 12 or 14 gauge cannula. Non-limiting examples of liposuctionprocedures include suction-assisted liposuction (SAL),ultrasound-assisted liposuction (UAL), power-assisted liposuction (PAL),twin-cannula (assisted) liposuction (TCAL or TCL), or externalultrasound-assisted liposuction (XUAL or EUAL), or water-assistedliposuction (WAL). In addition, the liposuction procedures listed abovecan be used with any of the following procedures that vary the amount offluid injected during the procedure, such as, e.g., dry liposuction, wetliposuction, super-wet liposuction, tumescent liposuction, orlaser-assisted liposuction. An autologous soft tissue transfer proceduretypically uses adipose tissue collected from a liposuction procedure.

Although the harvested tissue may be used directly to make the disclosedcompositions, it is more typically processed to purify and/or enrich forhealthy adipocytes and regenerative cells. For example, the harvestedadipose tissue may be separated from any debris and/or contaminants suchas, e.g., blood, serum, proteases, lipases, lipids and other oils,and/or other bodily fluids; tumescent fluid and/or other materials usedin the liposuction procedure; and/or other impurities suctioned duringthe procedure. Methods useful in separating debris and/or contaminantsfrom adipose tissue useful to make the disclosed compositions,including, without limitation, centrifugation, sedimentation,filtration, and/or absorption. In addition, or alternatively, theharvested adipose tissue may be processed by washing in a physiologicalbuffer like saline to remove any debris and/or contaminants.

A lipectomy procedure harvests adipose tissue by surgical excision froma donor site in a manner that minimizes damage to the blood supply ofthe tissue using standard surgical operative procedures. This harvestedtissue is then implanted into the region needing the soft tissuereplacement. A tissue flap or tissue graft procedure typically usesadipose tissue collected from a lipectomy procedure. A tissue flap is asection of living tissue that maintained its blood supply as the tissueis moved from one area of the body to another.

A local flap uses a piece of skin and underlying tissue that lieadjacent to the wound, including adipose tissue. The flap remainsattached at one end so that it continues to be nourished by its originalblood supply, and is repositioned over the wounded area. A regional flapuses a section of tissue that is attached by a specific blood vessel.When the flap is lifted, it needs only a very narrow attachment to theoriginal site to receive its nourishing blood supply from the tetheredartery and vein. A musculocutaneous flap, also called a muscle and skinflap, is used when the area to be covered needs more bulk and a morerobust blood supply. Musculocutaneous flaps are often used in breastreconstruction to rebuild a breast after mastectomy. As an example, thetransverse rectus abdominus myocutaneous flap (TRAM flap) is a tissueflap procedure that uses muscle, fat and skin from an abdomen to createa new breast mound after a mastectomy. This type of flap remains“tethered” to its original blood supply. In a bone/soft tissue flap,bone, along with the overlying skin, is transferred to the wounded area,carrying its own blood supply.

Typically, a wound that is wide and difficult or impossible to closedirectly may be treated with a skin graft. A skin graft is a patch ofhealthy skin that is taken from one area of the body, called the “donorsite,” and used to cover another area where skin is missing or damaged.There are three basic types of skin grafts. A split-thickness skingraft, commonly used to treat burn wounds, uses only the layers of skinclosest to the surface. A full-thickness skin graft might be used totreat a burn wound that is deep and large, or to cover jointed areaswhere maximum skin elasticity and movement are needed. As its nameimplies, a full-thickness (all layers) section of skin from the donorsite are lifted. A composite graft is used when the wound to be coveredneeds more underlying support, as with skin cancer on the nose. Acomposite graft requires lifting all the layers of skin, adipose tissue,and sometimes the underlying cartilage from the donor site.

The amount of adipose tissue collected will typically vary fromindividual to individual and can depend on a number of factorsincluding, but not limited to, amount of adipose tissue required for thesoft tissue replacement method, aesthetic expectations, age, bodyhabitus, coagulation profile, hemodynamic stability, co-morbidities, andphysician preference. A liposuction procedure may harvest from about 1mL to about 1500 mL of adipose tissue. A lipectomy procedure typicallyharvests about 1 g to about 5,000 g.

Adipose tissue comprises multiple regenerative cells. As used herein,the term “regenerative cell” refers to any cells that cause orcontribute to complete or partial regeneration, restoration, orsubstitution of structure or function of an organ, tissue, orphysiologic unit or system to thereby provide a therapeutic, structuralor cosmetic benefit. Examples of regenerative cells include stem cells,progenitor cells, and precursor cells. As used herein, the term “stemcell” refers to a multipotent regenerative cell with the potential todifferentiate into a variety of other cell types that perform one ormore specific functions and has the ability to self-renew. Some of thestem cells disclosed herein may be pluripotent. Exemplary examples ofstem cells include, without limitation, adipose-derived stem cells(ASCs; adipose-derived stromal cells), endothelial-derived stem cells(ESCs), hemopoietic stem cells (HSGs), and mesenchyma stem cells (MSCs).As used herein, the term “progenitor cell” refers to an oligopotentregenerative cell with the potential to differentiate into more than onecell type, or a unipotent regenerative cell with the potential todifferentiate into only a single cell type, that performs one or morespecific functions and has limited or no ability to self-renew.Exemplary examples of progenitor cells include, without limitation,endothelial progenitor cells, keratinocytes, monoblasts, myoblasts, andpericytes. As used herein, the term “precursor cell” refers to aunipotent regenerative cell with the potential to differentiate into onecell type that performs one or more specific functions and may retainextensive proliferative capacity that enables the cells to proliferateunder appropriate conditions. Exemplary examples of precursor cellsinclude, without limitation, adipoblast (lipoblast or preadipocytes),de-differentiated adipocytes, angioblasts, endothelial precursor cells,fibroblasts, lymphoblasts, and macrophages.

Harvested adipose tissue can be supplemented with regenerative cellssuch as, e.g., stem cells, progenitor cells, and precursor cells.Regenerative cells may promote new blood vessel formation, diminishnecrosis, and/or promote a supportive microenvironment in thetransplanted tissue, thereby improving survivability of the transplantedtissue. Regenerative cells can be obtained form a variety of sources.For example, adipose tissue is rich in regenerative cells that have theability to restore and reconstruct various soft tissue defects inresponse to local differentiation clues from the recipient site. Assuch, a portion of the collected adipose tissue may be further processedin order to purify regenerative cells that can then be added back to theremainder of the harvested adipose tissue in order to enrich thismaterial for these cells. Exemplary methods describing such cellenrichment procedures can be found in, e.g., U.S. Patent Publication2005/0025755, Yoshimura, et al., Characterization of Freshly Isolatedand Cultured Cells Derived form the Fatty and Fluid Portions ofliposuction Aspirates, J. Cell. Physiol. 208: 1011-1041 (2006);Yoshimura, et al., Cell-Assisted Lipotransfer for Facial Lipoatrophy:Effects of Clinical Use of Adipose-Derived Stem Cells, Dermatol. Surg.34: 1178-1185 (2008); Yoshimura, et al., Cell-Assisted Lipotransfer forCosmetic Breast Augmentation: Supportive Use of Adipose-DerivedStem/Stromal Cells, Aesth. Plast. Surg. 32: 48-55 (2008); each of whichis hereby incorporated by reference in its entirety.

In addition, harvested adipose tissue can be supplemented withregenerative cells obtained from cell cultures, such as, e.g., primarycell cultures and established cell cultures. For example, a portion ofharvested adipose tissue from an individual can be cultured in a mannerto produce primary cell cultures enriched for regenerative cells.Alternatively, established cell lines derived from regenerative cellsfrom adipose tissue, or another tissue source, can be cultured,harvested, and added to adipose tissue collected from an individual.Exemplary methods describing such cell culture compositions andprocedures can be found in, e.g., U.S. Patent Publication 2009/0246182;U.S. Patent Publication 2009/0317367; U.S. Patent Publication2008/0299213; Rehman, et al., Secretion of Angiogenic and AntiapoptoticFactors by Human Adipose Stromal Cells, Circulation, 109: r52-r58(2004); Kilroy, et al., Cytokine Profile of Human Adipose-Derived StemCells: Expression of Angiogenic, Hematopoietic, and Pro-InflammatoryFactors, J. Cell. Physiol., 212: 702-709 (2007); each of which is herebyincorporated by reference in its entirety.

Harvested adipose tissue may be immediately used to make thecompositions disclosed herein. Alternatively, harvested adipose tissue,whether unprocessed or processed, may be stored for use at some futuredate. Harvested tissue is typically stored using a slow freezing methodof the tissue to −20° C., with or without cryopreservatives. Storedadipose tissue can typically be stored for at least 6 months.

Aspects of the present specification provide, in part, a compositioncomprising a compound having the structure of formula I, apharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof,

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁₋₆ alkyl; R⁶is CO₂H, CO₂R⁷, CON(R⁷)₂, CONHCH₂CH₂OH, CON(CH₂CH₂OH)₂, CH₂OR⁷,P(O)(OR⁷)₂, or

R⁷ is H or C₁₋₆ hydrocarbyl; X and Y are each independently selectedfrom H, OH, ═O, Cl, Br, I, or CF₃; Z¹ and Z² are each independentlyselected from CH or N; W¹ and W² are each independently selected fromCH, CH₂, optionally substituted aryl, or optionally substitutedheteroaryl; m is 0, 1, 2, 3, 4, 5, or 6; o is 0, 1, 2, 3, 4; p is 0 or1; and V is C₁₋₆ alkyl, optionally substituted aryl, or optionallysubstituted heteroaryl.

The method of preparing the compounds represented by formula I can befound in, e.g., Donde, et el., 10,10-Dialkyl Prostanoic Acid Derivativesas Agents for Lowering Intraocular Pressure, U.S. Pat. No. 6,875,787;Donde, et el., 10,10-Dialkyl Prostanoic Acid Derivatives as Agents forLowering Intraocular Pressure, U.S. Patent Publication 2004/0235958;Donde, et al., Treatment of Inflammatory Bowel Disease, U.S. PatentPublication 2005/0164992, each of which is hereby incorporated byreference in its entirety. See also companion applications Jiang, etal., Compositions and Methods for Skin Repair, U.S. Provisional PatentApplication 61/374,439; and Jiang, et al., Compositions and Methods forTreating Corneal Haze, U.S. Provisional Patent Application 61/369,232;each of which is incorporated by reference in its entirety.

Unless otherwise indicated, any reference to a compound herein bystructure, name, or any other means, includes pharmaceuticallyacceptable salts, such as sodium, potassium, and ammonium salts;prodrugs, such as ester prodrugs; alternate solid forms, such aspolymorphs, solvates, hydrates, etc.; tautomers; or any other chemicalspecies that may rapidly convert to a compound described herein underconditions in which the compounds are used as described herein.

Unless stereochemistry is unambiguously depicted, any structure or namefor a compound used herein may refer to any stereoisomer or any mixtureof stereoisomers.

Unless otherwise indicated, when a compound or chemical structuralfeature such as aryl is referred to as being “optionally substituted,”it includes a feature that has no substituents (i.e. “unsubstituted”),or a feature that is “substituted,” meaning that the feature has one ormore substituents. The term “substituent” has the ordinary meaning knownto one of ordinary skill in the art, and includes a moiety that replacesone or more hydrogen atoms attached to a parent compound or structuralfeature. In some embodiments, the substituent may be an ordinary organicmoiety known in the art, which may have a molecular weight (e.g. the sumof the atomic masses of the atoms of the substituent) of 15 g/mol to 50g/mol, 15 g/mol to 100 g/mol, 15 g/mol to 150 g/mol, 15 g/mol to 200g/mol, 15 g/mol to 300 g/mol, or 15 g/mol to 500 g/mol. In someembodiments, the substituent comprises: 0-30, 0-20, 0-10, or 0-5 carbonatoms; and 0-30, 0-20, 0-10, or 0-5 heteroatoms independently selectedfrom: N, O, S, Si, F, Cl, Br, or I; provided that the substituentcomprises at least one atom selected from: C, N, O, S, Si, F, Cl, Br, orI. In some embodiments, a substituent may consist of 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, or 12 non-hydrogen atoms and any necessary hydrogenatoms. Some non-hydrogen atoms may include C, N, O, S, Si, F, Cl, Br, I,P, etc.

Examples of substituents include, but are not limited to, alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, aryl,heteroaryl, hydroxy, alkoxy, aryloxy, acyl, acyloxy, alkylcarboxylate,carboxylate, thiol, alkylthio, cyano, halo, thiocarbonyl, O-carbamyl,N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido,S-sulfonamido, N-sulfonamido, isocyanato, thiocyanato, isothiocyanato,nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxyl,trihalomethanesulfonyl, trihalomethanesulfonamido, amino, etc., or acombination thereof. The term “combination thereof” in the previous listindicates that substituents may also be a combination of any of theabove substituents, wherein a hydrogen atom of one substituent isreplaced by another substituent. For example, substituents may be acombination of alkyl and aryl (e.g. CH₂-phenyl, C₂H₄-heteraryl, etc.),alkyl and alkoxy (e.g. CH₂OCH₂), alkyl and halo (e.g. C₂H₄Cl, C₃H₆F,etc.), acyl and hydroxyl (e.g. —COCH₂OH), etc.

In some embodiments, a substituent may be R^(A), F, Cl, CN, OR^(A), CF₃,NO₂, NR^(A)R^(B), COR^(A), CO₂R^(A), OCOR^(A), NR^(A)COR^(B),CONR^(A)R^(B), etc.

Each R^(A) may independently be H, or C₁₋₁₂ alkyl, including: linear orbranched alkyl having a formula C_(a)H_(a+1), or cycloalkyl having aformula C_(a)H_(a−1), wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12, such as linear or branched alkyl of a formula: CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₁, C₆H₁₃, C₇H₁₅, C₈H₁₇, C₉H₁₉, C₁₀H₂₁, etc., or cycloalkyl ofa formula: C₃H₅, C₄H₇, C₅H₉, C₆H₁₁, C₇H₁₃, C₈H₁₅, C₉H₁₇, C₁₀H₁₉, etc.

Each R^(B) may independently be H, or C₁₋₁₂ alkyl, including: linear orbranched alkyl having a formula C_(a)H_(a+1), or cycloalkyl having aformula C_(a)H_(a), wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or12, such as linear or branched alkyl of a formula: CH₃, C₂H₅, C₃H₇,C₄H₉, C₅H₁₁, C₆H₁₃, C₈H₁₇, C₇H₁₅, C₉H₁₉, C₁₀H₂₁, etc., or cycloalkyl ofa formula: C₃H₅, C₄H₇, C₅H₉, C₆H₁₁, C₇H₁₃, C₈H₁₅, C₉H₁₇, C₁₀H₁₉, etc.

For convenience, the term “molecular weight” is used with respect to amoiety or part of a molecule to indicate the sum of the atomic masses ofthe atoms in the moiety or part of a molecule, even though it may not bea complete molecule.

Structures associated with some of the chemical names referred to hereinare depicted below. These structures may be unsubstituted, as shownbelow, or a substituent may independently be in any position normallyoccupied by a hydrogen atom when the structure is unsubstituted. Unlessa point of attachment is indicated by

attachment may occur at any position normally occupied by a hydrogenatom.

As used herein, the term “hydrocarbyl” has the broadest meaninggenerally understood in the art, and may include a moiety composed ofcarbon and hydrogen. Some examples may include alkyl, alkenyl, alkynyl,aryl, etc., and combinations thereof, and may be linear, branched,cyclic, or a combination thereof. Hydrocarbyl may be bonded to any othernumber of moieties (e.g. be bonded to 1 other group, such as —CH₃,—CH═CH₂, etc.; 2 other groups, such as -phenyl-, —C≡C—, etc.; or anynumber of other groups) that the structure may bear, and in someembodiments, may contain from one to thirty-five carbon atoms. Examplesof hydrocarbyl groups include but are not limited to C₁ alkyl, C₂ alkyl,C₂ alkenyl, C₂ alkynyl, C₃ alkyl, C₃ alkenyl, C₃ alkynyl, C₄ alkyl, C₄alkenyl, C₄ alkynyl, C₅ alkyl, C₅ alkenyl, C₅ alkynyl, C₆ alkyl, C₆alkenyl, C₆ alkynyl, phenyl, etc.

As used herein the term “alkyl” has the broadest meaning generallyunderstood in the art, and may include a moiety composed of carbon andhydrogen containing no double or triple bonds. Alkyl may be linearalkyl, branched alkyl, cycloalkyl, or a combination thereof, and in someembodiments, may contain from one to thirty-five carbon atoms. In someembodiments, alkyl may include C₁₋₁₀ linear alkyl, such as methyl(—CH₃), ethyl (—CH₂CH₃), n-propyl (—CH₂CH₂CH₃), n-butyl (—CH₂CH₂CH₂CH₃),n-pentyl (—CH₂CH₂CH₂CH₂CH₃), n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), etc.; C₃₋₁₀branched alkyl, such as C₃H₇ (e.g. iso-propyl), C₄H₉ (e.g. branchedbutyl isomers), C₅H₁₁ (e.g. branched pentyl isomers), C₆H₁₃ (e.g.branched hexyl isomers), C₇H₁₅ (e.g. heptyl isomers), etc.; C₃₋₁₀cycloalkyl, such as C₃H₅ (e.g. cyclopropyl), C₄H₇ (e.g. cyclobutylisomers such as cyclobutyl, methylcyclopropyl, etc.), C₅H₉ (e.g.cyclopentyl isomers such as cyclopentyl, methylcyclobutyl,dimethylcyclopropyl, etc.) C₆H₁₁ (e.g. cyclohexyl isomers), C₇H₁₃ (e.g.cycloheptyl isomers), etc.; and the like.

As used herein, the term “heterocyclyl” has the broadest meaninggenerally understood in the art, and may include a heterocyclic ring, ora ring containing one or more heteroatom as ring atoms.

As used herein the term “aryl” has the broadest meaning generallyunderstood in the art, and may include an aromatic ring or aromatic ringsystem such as phenyl, naphthyl, etc. Any aryl identified herein may beC₆₋₁₀ aryl. The term “heteroaryl” also has the meaning understood by aperson of ordinary skill in the art, and may refer to an “aryl” whichhas one or more heteroatoms in the ring or ring system. Examples of“heteroaryl” may include, but are not limited to, pyridinyl, furyl,thienyl, oxazolyl, thiazolyl, imidazolyl, indolyl, quinolinyl,benzofuranyl, benzothienyl, benzooxazolyl, benzothiazolyl,benzoimidazolyl, etc. Any heteroaryl identified herein may be C₃₋₉heteroaryl.

As used herein, the term “haloalkyl” refers to alkyl having one or morehalo substituents.

In another embodiment, the compound has the structure of formula II,

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁₋₆ alkyl; R⁴is H or C₁₋₆ hydrocarbyl; X and Y are each independently selected fromH, OH, ═O, Cl, Br, I, or CF₃; Z¹ and Z² are each independently selectedfrom CH or N; W¹ and W² are each independently selected from CH, CH₂,optionally substituted aryl, or optionally substituted heteroaryl; m is0, 1, 2, 3, or 4; o is 0, 1, 2, 3, or 4; p is 0 or 1; and V is CH₃,aryl, optionally substituted aryl, or optionally substituted heteroaryl.

In an aspect of this embodiment, V is

wherein U is N, O, or S; R⁵ is F, Cl, Br, I, C₁₋₆ hydrocarbyl; and n is0, 1, 2, 3, 4, 5, 6, or 7; and. In another aspect of this embodiment, Uis S; R⁵ is F, Cl, Br, or I; and n is 1, 2, or 3. In yet another aspectof this embodiment W² is thienyl.

In another embodiment, the compound has the structure of formula III

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁₋₆ alkyl; R⁴is H or C₁-C₆ hydrocarbyl; W¹ and W² are each independently selectedfrom CH, CH₂, optionally substituted aryl, or optionally substitutedheteroaryl; m is 0, 1, 2, 3, or 4; o is 0, 1, 2, 3, or 4; p is 0 or 1;and V is CH₃, optionally substituted aryl or optionally substitutedheteroaryl.

In yet another embodiment, the compound has the structure of formula IV:

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁₋₆ linearalkyl; R⁴ is H, C₁₋₆ hydrocarbyl; m is 0, 1, 2, 3, or 4; o is 0, 1, 2,3, or 4; p is 0 or 1; and V is CH₃, optionally substituted aryl oroptionally substituted heteroaryl.

In still another embodiment, the compound has the structure of formulaV:

wherein each dashed line represents the presence or absence of a bond;R⁴ is H, C₁₋₆ hydrocarbyl; o is 0, 1, 2, 3, or 4; and V is CH₃,optionally substituted aryl or optionally substituted heteroaryl.

In a further embodiment, the compound has a structure of one of thefollowing compounds listed in Table 1.

TABLE 1 Low Rf High Rf Structure diastereomer diastereomer

34 35

36 37

38 39

40 41

42

43

44

45

46 47

48 49

50 51

52 53

54 55

56 57

58 59

60 61

62 63

64 65

66 67

68 69

70 71

72 73

74 75

In a further embodiment, the compound has a structure of compound 1, 2,3, 4, or 5.

In a yet further embodiment, the compound has a structure of compound 6.

Other aspects of the present specification provide, in part, acomposition comprising a compound having the structure of formula VI

wherein each dashed line represents the presence or absence of a bond;Z² and Z³ are independently CH or N; Z¹, Z⁴, Z⁵, and Z⁶ are eachindependently CR⁹, CHR⁹, NH, O, or S; A is —(CH₂)₆—, or cis—CH₂CH═CH—(CH₂)₃—, wherein 1 or 2 carbons may be substituted with S orO; or A is —(CH₂)_(m)—Ar—(CH₂)_(o)— wherein Ar is optionally substitutedaryl or optionally substituted heteroaryl, the sum of m and o is from 1,2, 3, or 4, and wherein one CH₂ may be substituted with S or O; R⁸ is H,hydrocarbyl, or hydrocarbyl-OH; R⁹ is independently H, C₁₋₆ alkyl, OH,F, Cl, Br, I, or O; J is C₁₋₆ alkyl, C₁₋₆—O-alkyl, C═O, or CHOH; E isC₁₋₁₂ alkyl, R¹⁰, or —Y—R¹⁰ wherein Y is CH₂, S, or O, and R¹⁰ isoptionally substituted aryl or optionally substituted heteroaryl; and nis 0 or 1.

Methods of preparing the compounds represented by formula VI can befound in, e.g., Maruyama and Ohuchida, 5-Thia-ω-SubstitutedPhenyl-Prostaglandin E Derivatives, Process for Producing the Same andDrugs Containing the Same as the Active Ingredient, U.S. Pat. No.6,462,081; Cameron and Lefker, EP4 Receptor Selective Agonists in theTreatment of Osteoporosis, U.S. Pat. No. 6,552,067; Maruyama, et al.,Pharmaceutical Composition for Treatment of Diseases Associated withDecrease in Bone Mass Comprising EP4 Agonist as the Active Ingredient,U.S. Pat. No. 7,608,637; Ogidigben, et al., Method for Treating OcularHypertension, U.S. Patent Publication 2004/0254230; Kuwahara, et al.,Preventive and/or Remedy for Hyperkalemia Containing EP4 Agonist, U.S.Patent Publication 2008/0234337; Takigawa, et al., Cytotoxic T CellActivator Comprising EP4 Agonist, U.S. Patent Publication 2010/0216689;and Ono, et al., Prostaglandin Derivative, WO 2006/137472; each of whichis incorporated by reference in its entirety.

In another embodiment, the compound has the structure of formula VII,

wherein each dashed line represents the presence or absence of a bond;Z² is CH or N; Z⁵ is CH₂, NH, O, or S; Z⁷ is CH₂, O, or S; R⁸ is H, C₁₋₆hydrocarbyl, or C₁₋₆ hydrocarbyl-OH; R⁹ and R¹¹ are independently C₁₋₆alkyl, OH, F, Cl, Br, I, or O; and R¹² is H, OH, O, F, Cl, Br, I,C₁₋₄—O-alkyl, C₁₋₄ haloalkyl, or aryl.

In another embodiment, the compound has the structure of formula VIII,

wherein each dashed line represents the presence or absence of a bond;Z² is CH or N; and Z⁵ is CH₂, NH, O, or S; Z⁷ and Z⁸ are independentlyCH₂, O, or S; R⁸ is H, C₁₋₆ hydrocarbyl, or C₁₋₆ alkyl-OH; R⁹ and R¹¹are independently C₁₋₆ alkyl, OH, F, Cl, Br, I, or O; and R¹² is H, OH,O, F, Cl, Br, I, C₁₋₄—O-alkyl, C₁₋₄ haloalkyl, or aryl.

In an aspect of this embodiment, the compound has a structure ofcompound 6, 7, 8, 9, 10, 11, 12, or 13.

Other aspects of the present specification provide, in part, acomposition comprising a compound having the structure of formula IX

wherein X¹ and X⁵ are independently CH or N; X⁷ is NH, O, or S; X⁸ andX⁹ are independently CH or N; R¹³ is optionally substituted aryl oroptionally substituted heteroaryl; and R¹⁴ is C₁₋₆ alkyl, optionallysubstituted aryl, or optionally substituted heteroaryl.

Methods of preparing the compounds represented by formula IX can befound in, e.g., Linders, et al., Bicyclic Derivatives as EP4 Agonists,WO 2008/092860; Linders, et al., Bicyclic Derivatives as EP4 Agonists,WO 2008/092861; and Linders, et al., Bicyclic Derivatives as EP4Agonists, WO 2008/092862; each of which is incorporated by reference inits entirety.

In another embodiment, the compound has the structure of formula X,

wherein X¹⁰, X¹², X¹³, X¹⁴, and X¹⁵ are each independently CR¹⁹ or N;R¹⁶ is independently H, OH, O, F, Cl, Br, I, C₁₋₆ alkyl, C₁₋₆—O-alkyl,CN, or C₁₋₆ alkyl-CN; and R¹⁹ is independently H, OH, O, F, Cl, Br, I,C₁₋₁₀ alkyl, C₁₋₁₀—O-alkyl, or C₄₋₁₀—O-alkyl-heterocyclyl. In an aspectof this embodiment, the C₄₋₁₀ O-alkyl-heterocyclyl is

In another embodiment, the compound has the structure of formula XI,

wherein X¹⁴ is CR¹⁹ or N; R¹⁶, R¹⁷, and R¹⁸ are each independently H,OH, O, F, Cl, Br, I, C₁₋₆ alkyl, C₁₋₆—O-alkyl, CN, or C₁₋₆ alkyl-CN; R¹⁹is H, OH, O, or F, Cl, Br, I, C₁₋₁₀ alkyl, C₁₋₁₀—O-alkyl, or

andR²⁰ is H, F, Cl, Br, or I.

In another embodiment, the compound has a structure of compound 14, 15,16, 17, 18, 19, 20, 21, 22, or 23, a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable amine salt thereof.

Other aspects of the present specification provide, in part, acomposition comprising a compound having the structure of compound 24, apharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof. In aspects of this embodiment, apharmaceutically acceptable salt is a sodium salt and a potassium salt.In other aspects of this embodiment, a pharmaceutically acceptable aminesalt is an aminoglycoside salt and a triethanolamine salt.

Methods of preparing the compounds represented by compound 24 can befound in, e.g., Keel and Vallance, Novel Salts of(4-{[(5-{[(3-Chlorophenyl)Methyl]Oxy}-2-Methylphenyl)Carbonyl]Amino}-3-Methylphenyl)AceticAcid, U.S. patent application Ser. No. 12/997,062 filed Dec. 9, 2010 isa national phase application of WO 2009/150118 (PCT/EP09/57007) filed onJun. 8, 2009; which is incorporated by reference in its entirety.

The compositions disclosed herein may optionally comprise any number andcombination of compounds disclosed herein. For instance, a compositioncan comprise, e.g., two or more compounds, three or more compounds, fouror more compounds or five or more compounds.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising a glycosaminoglycan polymer. The hydrogel materialdisclosed herein can further comprise two or more differentglycosaminoglycan polymers. As used herein, the term “glycosaminoglycan”is synonymous with “GAG” and “mucopolysaccharide” and refers to longunbranched polysaccharides consisting of a repeating disaccharide units.The repeating unit consists of a hexose (six-carbon sugar) or ahexuronic acid, linked to a hexosamine (six-carbon sugar containingnitrogen) and pharmaceutically acceptable salts thereof. Members of theGAG family vary in the type of hexosamine, hexose or hexuronic acid unitthey contain, such as, e.g., glucuronic acid, iduronic acid, galactose,galactosamine, glucosamine) and may also vary in the geometry of theglycosidic linkage. Any glycosaminoglycan polymer is useful in thehydrogel materials disclosed herein with the proviso that theglycosaminoglycan polymer improves a condition of the skin. Non-limitingexamples of glycosaminoglycans include chondroitin sulfate, dermatansulfate, keratan sulfate, hyaluronan. Non-limiting examples of anacceptable salt of a glycosaminoglycans includes sodium salts, potassiumsalts, magnesium salts, calcium salts, and combinations thereof.Glycosaminoglycan and their resulting polymers useful in the hydrogelmaterials and methods disclosed herein are described in, e.g., Piron andTholin, Polysaccharide Crosslinking, Hydrogel Preparation, ResultingPolysaccharides(s) and Hydrogel(s), uses Thereof, U.S. PatentPublication 2003/0148995; Lebreton, Cross-Linking of Low and HighMolecular Weight Polysaccharides Preparation of Injectable MonophaseHydrogels; Lebreton, Viscoelastic Solutions Containing SodiumHyaluronate and Hydroxypropyl Methyl Cellulose, Preparation and Uses,U.S. Patent Publication 2008/0089918; Lebreton, Hyaluronic Acid-BasedGels Including Lidocaine, U.S. Patent Publication 2010/0028438; andPolysaccharides and Hydrogels thus Obtained, U.S. Patent Publication2006/0194758; and Di Napoli, Composition and Method for Intradermal SoftTissue Augmentation, International Patent Publication WO 2004/073759,each of which is hereby incorporated by reference in its entirety. GAGsuseful in the hydrogel materials and methods disclosed herein arecommercially available, such as, e.g., hyaluronan-based dermal fillersJUVEDERM®, JUVEDERM® 30, JUVEDERM® Ultra, JUVEDERM® Ultra Plus,JUVEDERM® Ultra XC, and JUVEDERM® Ultra Plus XC (Allergan Inc, Irvine,Calif.). Table 2 lists representative GAGs.

TABLE 2 Examples of GAGs Glycosidic Hexuronic linkage Name acid/HexoseHexosamine geometry Unique features Chondroitin GlcUA or GalNAc or-4GlcUAβ1- Most prevalent GAG sulfate GlcUA(2S) GalNAc(4S) or 3GalNAcβ1-GalNAc(6S) or GalNAc(4S,6S) Dermatan GlcUA or GalNAc or -4IdoUAβ1-Distinguished from chondroitin sulfate IdoUA or GalNAc(4S) or 3GalNAcβ1-sulfate by the presence of iduronic IdoUA(2S) GalNAc(6S) or acid,although some hexuronic GalNAc(4S,6S) acid monosaccharides may beglucuronic acid. Keratan Gal or GlcNAc or -3Gal(6S)β1- Keratan sulfatetype II may be sulfate Gal(6S) GlcNAc(6S) 4GlcNAc(6S)β1- fucosylated.Heparin GlcUA or GlcNAc or -4IdoUA(2S)α1- Highest negative chargedensity of IdoUA(2S) GlcNS or 4GlcNS(6S)α1- any known biologicalmolecule GlcNAc(6S) or GlcNS(6S) Heparan GlcUA or GlcNAc or -4GlcUAβ1-Highly similar in structure to sulfate IdoUA or GlcNS or 4GlcNAcα1-heparin, however heparan sulfates IdoUA(2S) GlcNAc(6S) or disaccharideunits are organised GlcNS(6S) into distinct sulfated and non- sulfateddomains. Hyaluronan GlcUA GlcNAc -4GlcUAβ1- The only GAG that isexclusively 3GlcNAcβ1- non-sulfated GlcUA = β-D-glucuronic acidGlcUA(2S) = 2-O-sulfo-β-D-glucuronic acid IdoUA = α-L-iduronic acidIdoUA(2S) = 2-O-sulfo-α-L-iduronic acid Gal = β-D-galactose Gal(6S) =6-O-sulfo-β-D-galactose GalNAc = β-D-N-acetylgalactosamine GalNAc(4S) =β-D-N-acetylgalactosamine-4-O-sulfate GalNAc(6S) =β-D-N-acetylgalactosamine-6-O-sulfate GalNAc(4S,6S) =β-D-N-acetylgalactosamine-4-O, 6-O-sulfate GlcNAc =α-D-N-acetylglucosamine GlcNS = α-D-N-sulfoglucosamine GlcNS(6S) =α-D-N-sulfoglucosamine-6-O-sulfate

Aspects of the present specification provide, in part, a hydrogelmaterial comprising a chondroitin sulfate polymer. As used herein, theterm “chondroitin sulfate polymer” refers to an unbranched, sulfatedpolymer of variable length comprising disaccharides of two alternatingmonosaccharides of D-glucuronic acid (GlcA) and N-acetyl-D-galactosamine(GalNAc) and pharmaceutically acceptable salts thereof. A chondroitinsulfate polymer may also include D-glucuronic acid residues that areepimerized into L-iduronic acid (IdoA), in which case the resultingdisaccharide is referred to as dermatan sulfate. A chondroitin sulfatepolymer can have a chain of over 100 individual sugars, each of whichcan be sulfated in variable positions and quantities. Chondroitinsulfate polymers are an important structural component of cartilage andprovide much of its resistance to compression. Any chondroitin sulfatepolymer is useful in the hydrogel materials disclosed herein with theproviso that the chondroitin sulfate polymer improves a condition of theskin. Non-limiting examples of pharmaceutically acceptable salts ofchondroitin sulfate include sodium chondroitin sulfate, potassiumchondroitin sulfate, magnesium chondroitin sulfate, calcium chondroitinsulfate, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising a keratan sulfate polymer. As used herein, the term“keratan sulfate polymer” refers to a polymer of variable lengthcomprising disaccharide units, which themselves include β-D-galactoseand N-acetyl-D-galactosamine (GalNAc) and pharmaceutically acceptablesalts thereof. Disaccharides within the repeating region of keratansulfate may be fucosylated and N-Acetylneuraminic acid caps the end ofthe chains. Any keratan sulfate polymer is useful in the hydrogelmaterials disclosed herein with the proviso that the keratan sulfatepolymer improves a condition of the skin. Non-limiting examples ofpharmaceutically acceptable salts of keratan sulfate include sodiumkeratan sulfate, potassium keratan sulfate, magnesium keratan sulfate,calcium keratan sulfate, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising a hyaluronan polymer. As used herein, the term“hyaluronic acid polymer” is synonymous with “HA polymer”, “hyaluronicacid polymer”, and “hyaluronate polymer” refers to an anionic,non-sulfated glycosaminoglycan polymer comprising disaccharide units,which themselves include D-glucuronic acid and D-N-acetylglucosaminemonomers, linked together via alternating β-1,4 and β-1,3 glycosidicbonds and pharmaceutically acceptable salts thereof. Hyaluronan polymerscan be purified from animal and non-animal sources. Polymers ofhyaluronan can range in size from about 5,000 Da to about 20,000,000 Da.Any hyaluronan polymer is useful in the hydrogel materials disclosedherein with the proviso that the hyaluronan improves a condition of theskin. Non-limiting examples of pharmaceutically acceptable salts ofhyaluronan include sodium hyaluronan, potassium hyaluronan, magnesiumhyaluronan, calcium hyaluronan, and combinations thereof.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising a crosslinked glycosaminoglycan polymer. As usedherein, the term “crosslinked” refers to the intermolecular bondsjoining the individual polymer molecules, or monomer chains, into a morestable structure like a gel. As such, a crosslinked glycosaminoglycanpolymer has at least one intermolecular bond joining at least oneindividual polymer molecule to another one. The crosslinking ofglycosaminoglycan polymers typically result in the formation of ahydrogel. Such hydrogels have high viscosity and require considerableforce to extrude through a fine needle. Glycosaminoglycan polymersdisclosed herein may be crosslinked using dialdehydes and disulfidescrosslinking agents including, without limitation, multifunctionalPEG-based crosslinking agents, divinyl sulfones, diglycidyl ethers, andbis-epoxides. Non-limiting examples of hyaluronan crosslinking agentsinclude multifunctional PEG-based crosslinking agents likepentaerythritol tetraglycidyl ether (PETGE), divinyl sulfone (DVS),1,4-butanediol diglycidyl ether (BDDE),1,2-bis(2,3-epoxypropoxy)ethylene (EGDGE), 1,2,7,8-diepoxyoctane (DEO),biscarbodiimide (BCD), adipic dihydrazide (ADH),bis(sulfosuccinimidyl)suberate (BS), hexamethylenediamine (NMDA),1-(2,3-epoxypropyl)-2,3-epoxycyclohexane, or combinations thereof. Otheruseful cross-linking agents are disclosed in Stroumpoulis and Tezel,Tunably Crosslinked Polysaccharide Compositions, U.S. patent applicationSer. No. 12/910,466, filed Oct. 22, 2010, which is incorporated byreference in its entirety. Non-limiting examples of methods ofcrosslinking glycosaminoglycan polymers are described in, e.g.,Glycosaminoglycan polymers useful in the hydrogel materials and methodsdisclosed herein are described in, e.g., Piron and Tholin,Polysaccharide Crosslinking, Hydrogel Preparation, ResultingPolysaccharides(s) and Hydrogel(s), uses Thereof, U.S. PatentPublication 2003/0148995; Lebreton, Cross-Linking of Low and HighMolecular Weight Polysaccharides Preparation of Injectable MonophaseHydrogels and Polysaccharides and Hydrogels thus Obtained, U.S. PatentPublication 2006/0194758; Lebreton, Viscoelastic Solutions ContainingSodium Hyaluronate and Hydroxypropyl Methyl Cellulose, Preparation andUses, U.S. Patent Publication 2008/0089918; Lebreton, HyaluronicAcid-Based Gels Including Lidocaine, U.S. Patent Publication2010/0028438; and Di Napoli, Composition and Method for Intradermal SoftTissue Augmentation, International Patent Publication WO 2004/073759,each of which is hereby incorporated by reference in its entirety.

In an embodiment, a hydrogel material comprises a crosslinkedglycosaminoglycan polymer where the crosslinked glycosaminoglycanpolymer is present in an amount sufficient to improve a skin conditionas disclosed herein. In aspect of this embodiment, a hydrogel materialcomprises a crosslinked chondroitin sulfate polymer, a crosslinkeddermatan sulfate polymer, a crosslinked keratan sulfate polymer, acrosslinked heparan polymer, a crosslinked heparan sulfate polymer, or acrosslinked hyaluronan polymer. In other aspects of this embodiment, ahydrogel material comprises a crosslinked glycosaminoglycan where thecrosslinked glycosaminoglycan represents, e.g., about 1% by weight,about 2% by weight, about 3% by weight, about 4% by weight, about 5% byweight, about 6% by weight, about 7% by weight, about 8% by weight, orabout 9%, or about 10% by weight, of the total glycosaminoglycan presentin the hydrogel material. In yet other aspects of this embodiment, ahydrogel material comprises a crosslinked glycosaminoglycan where thecrosslinked glycosaminoglycan represents, e.g., at most 1% by weight, atmost 2% by weight, at most 3% by weight, at most 4% by weight, at most5% by weight, at most 6% by weight, at most 7% by weight, at most 8% byweight, at most 9% by weight, or at most 10% by weight, of the totalglycosaminoglycan present in the hydrogel material. In still otheraspects of this embodiment, a hydrogel material comprises a crosslinkedglycosaminoglycan where the crosslinked glycosaminoglycan represents,e.g., about 0% to about 10% by weight, about 1% to about 10% by weight,about 3% to about 10% by weight, or about 5% to about 10% by weight, ofthe total glycosaminoglycan present in the hydrogel material.

In aspects of this embodiment, a hydrogel material comprises acrosslinked glycosaminoglycan where the crosslinked glycosaminoglycan ispresent at a concentration of, e.g., about 2 mg/mL, about 3 mg/mL, about4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7 mg/mL, about 8 mg/mL,about 9 mg/mL, about 10 mg/mL, about 11 mg/mL, about 12 mg/mL, about 13mg/mL, about 13.5 mg/mL, about 14 mg/mL, about 15 mg/mL, about 16 mg/mL,about 17 mg/mL, about 18 mg/mL, about 19 mg/mL, or about 20 mg/mL. Inother aspects of this embodiment, a hydrogel material comprises acrosslinked glycosaminoglycan where the crosslinked glycosaminoglycan ispresent at a concentration of, e.g., at least 1 mg/mL, at least 2 mg/mL,at least 3 mg/mL, at least 4 mg/mL, at least 5 mg/mL, at least 10 mg/mL,at least 15 mg/mL, at least 20 mg/mL, or at least 25 mg/mL. In yet otheraspects of this embodiment, a hydrogel material comprises a crosslinkedglycosaminoglycan where the crosslinked glycosaminoglycan is present ata concentration of, e.g., at most 1 mg/mL, at most 2 mg/mL, at most 3mg/mL, at most 4 mg/mL, at most 5 mg/mL, at most 10 mg/mL, at most 15mg/mL, at most 20 mg/mL, or at most 25 mg/mL. In still other aspects ofthis embodiment, a hydrogel material comprises a crosslinkedglycosaminoglycan where the crosslinked glycosaminoglycan is present ata concentration of, e.g., about 7.5 mg/mL to about 19.5 mg/mL, about 8.5mg/mL to about 18.5 mg/mL, about 9.5 mg/mL to about 17.5 mg/mL, about10.5 mg/mL to about 16.5 mg/mL, about 11.5 mg/mL to about 15.5 mg/mL, orabout 12.5 mg/mL to about 14.5 mg/mL.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising hyaluronan polymers of low molecular weight,hyaluronan polymers of high molecular weight, or hyaluronan polymers ofboth low and high molecular weight. As used herein, the term “highmolecular weight” when referring to “hyaluronan” refers to hyaluronanpolymers having a mean molecular weight of 1,000,000 Da or greater.Non-limiting examples of a high molecular weight hyaluronan polymersinclude hyaluronan polymers about 1,500,000 Da, about 2,000,000 Da,about 2,500,000 Da, about 3,000,000 Da, about 3,500,000 Da, about4,000,000 Da, about 4,500,000 Da, and about 5,000,000 Da. As usedherein, the term “low molecular weight” when referring to “hyaluronan”refers to hyaluronan polymers having a mean molecular weight of lessthan 1,000,000 Da. Non-limiting examples of a low molecular weighthyaluronan polymers include hyaluronan polymers of about 200,000 Da,about 300,000 Da, about 400,000 Da, about 500,000 Da, about 600,000 Da,about 700,000 Da, of about 800,000 Da, and about 900,000 Da.

In an embodiment, a hydrogel material comprises crosslinked hyaluronanpolymers of low molecular weight. In aspects of this embodiment, ahydrogel material comprises crosslinked hyaluronan polymers having amean molecular weight of, e.g., about 100,000 Da, about 200,000 Da,about 300,000 Da, about 400,000 Da, about 500,000 Da, about 600,000 Da,about 700,000 Da, about 800,000 Da, or about 900,000 Da. In yet otheraspects of this embodiment, a hydrogel material comprises crosslinkedhyaluronan polymers having a mean molecular weight of, e.g., at most100,000 Da, at most 200,000 Da, at most 300,000 Da, at most 400,000 Da,at most 500,000 Da, at most 600,000 Da, at most 700,000 Da, at most800,000 Da, at most 900,000 Da, or at most 950,000. In still otheraspects of this embodiment, a hydrogel material comprises crosslinkedhyaluronan polymers having a mean molecular weight of, e.g., about100,000 Da to about 500,000 Da, about 200,000 Da to about 500,000 Da,about 300,000 Da to about 500,000 Da, about 400,000 Da to about 500,000Da, about 500,000 Da to about 950,000 Da, about 600,000 Da to about950,000 Da, about 700,000 Da to about 950,000 Da, about 800,000 Da toabout 950,000 Da, about 300,000 Da to about 600,000 Da, about 300,000 Dato about 700,000 Da, about 300,000 Da to about 800,000 Da, or about400,000 Da to about 700,000 Da.

In another embodiment, a hydrogel material comprises crosslinkedhyaluronan polymers of high molecular weight. In aspects of thisembodiment, a hydrogel material comprises a crosslinked hyaluronanpolymers having a mean molecular weight of, e.g., about 1,000,000 Da,about 1,500,000 Da, about 2,000,000 Da, about 2,500,000 Da, about3,000,000 Da, about 3,500,000 Da, about 4,000,000 Da, about 4,500,000Da, or about 5,000,000 Da. In yet other aspects of this embodiment, ahydrogel material comprises a crosslinked hyaluronan polymers having amean molecular weight of, e.g., at least 1,000,000 Da, at least1,500,000 Da, at least 2,000,000 Da, at least 2,500,000 Da, at least3,000,000 Da, at least 3,500,000 Da, at least 4,000,000 Da, at least4,500,000 Da, or at least 5,000,000 Da. In still other aspects of thisembodiment, a hydrogel material comprises a crosslinked hyaluronanpolymers having a mean molecular weight of, e.g., about 1,000,000 Da toabout 5,000,000 Da, about 1,500,000 Da to about 5,000,000 Da, about2,000,000 Da to about 5,000,000 Da, about 2,500,000 Da to about5,000,000 Da, about 2,000,000 Da to about 3,000,000 Da, about 2,500,000Da to about 3,500,000 Da, or about 2,000,000 Da to about 4,000,000 Da.

In yet another embodiment, a hydrogel material comprises a crosslinkedhyaluronan polymers where the crosslinked hyaluronan polymers comprise acombination of both high molecular weight hyaluronan polymers and lowmolecular weight hyaluronan polymers, in various ratios. In aspects ofthis embodiment, a hydrogel material comprises a crosslinked hyaluronanpolymers where the crosslinked hyaluronan polymers comprises acombination of both high molecular weight hyaluronan polymers and lowmolecular weight hyaluronan polymers in a ratio of about 20:1, about15:1, about 10:1, about 5:1, about 1:1, about 1:5 about 1:10, about1:15, or about 1:20.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising a crosslinked glycosaminoglycan polymer having adegree of crosslinking. As used herein, the term “degree ofcrosslinking” refers to the percentage of glycosaminoglycan polymermonomeric units, such as, e.g., the disaccharide monomer units ofhyaluronan that are bound to a cross-linking agent. The degree ofcrosslinking is expressed as the percent weight ratio of thecrosslinking agent to glycosaminoglycan monomeric unit within thecrosslinked portion of the hydrogel material. It is measured by theweight ratio of glycosaminoglycan monomers to crosslinker. Thus, ahydrogel material that that has a crosslinked glycosaminoglycan polymerwith a 4% degree of crosslinking means that on average there are fourcrosslinking molecules for every 100 glycosaminoglycan monomeric units.Every other parameter being equal, the greater the degree ofcrosslinking, the harder the hydrogel becomes. Non-limiting examples ofa degree of crosslinking useful to make the hydrogel materials disclosedherein include about 1% to about 15%.

In aspects of this embodiment, a hydrogel material comprises acrosslinked glycosaminoglycan where the degree of crosslinking is about1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, orabout 15%. In yet other aspects of this embodiment, a hydrogel materialcomprises a crosslinked glycosaminoglycan where the degree ofcrosslinking is at most 1%, at most 2%, at most 3%, at most 4%, at most5%, at most 6%, at most 7%, at most 8%, at most 9%, at most 10%, at most11%, at most 12%, at most 13%, at most 14%, or at most 15%. In stillother aspects of this embodiment, a hydrogel material comprises acrosslinked glycosaminoglycan where the degree of crosslinking is about1% to about 15%, about 2% to about 11%, about 3% to about 10%, about 1%to about 5%, about 10% to about 15%, about 11% to about 15%, about 6% toabout 10%, or about 6% to about 8%.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising an uncrosslinked glycosaminoglycan polymer. As usedherein, the term “uncrosslinked” refers to a lack of intermolecularbonds joining the individual glycosaminoglycan polymer molecules, ormonomer chains. As such, an uncrosslinked glycosaminoglycan polymer isnot linked to any other glycosaminoglycan polymer by an intermolecularbond. In aspects of this embodiment, a hydrogel material comprises anuncrosslinked chondroitin sulfate polymer, an uncrosslinked dermatansulfate polymer, an uncrosslinked keratan sulfate polymer, anuncrosslinked heparan polymer, an uncrosslinked heparan sulfate polymer,or an uncrosslinked hyaluronan polymer. Uncrosslinked glycosaminoglycanpolymers are water soluble and generally remain fluid in nature. Assuch, uncross-linked glycosaminoglycan polymers are often mixed with aglycosaminoglycan polymer-based hydrogel material as a lubricant tofacilitate the extrusion process of the hydrogel material through a fineneedle.

In an embodiment, a hydrogel material comprises an uncrosslinkedglycosaminoglycan polymer where the uncrosslinked glycosaminoglycanpolymer is present in an amount sufficient to improve a skin conditionas disclosed herein. In aspects of this embodiment, a hydrogel materialcomprises an uncrosslinked glycosaminoglycan where the uncrosslinkedglycosaminoglycan is present at a concentration of, e.g., about 2 mg/mL,about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6 mg/mL, about 7mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL, about 11 mg/mL,about 12 mg/mL, about 13 mg/mL, about 13.5 mg/mL, about 14 mg/mL, about15 mg/mL, about 16 mg/mL, about 17 mg/mL, about 18 mg/mL, about 19mg/mL, about 20 mg/mL, about 40 mg/mL, or about 60 mg/mL. In otheraspects of this embodiment, a hydrogel material comprises anuncrosslinked glycosaminoglycan where the uncrosslinkedglycosaminoglycan is present at a concentration of, e.g., at least 1mg/mL, at least 2 mg/mL, at least 3 mg/mL, at least 4 mg/mL, at least 5mg/mL, at least 10 mg/mL, at least 15 mg/mL, at least 20 mg/mL, at least25 mg/mL at least 35 mg/mL, or at least 40 mg/mL. In yet other aspectsof this embodiment, a hydrogel material comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan is presentat a concentration of, e.g., at most 1 mg/mL, at most 2 mg/mL, at most 3mg/mL, at most 4 mg/mL, at most 5 mg/mL, at most 10 mg/mL, at most 15mg/mL, at most 20 mg/mL, or at most 25 mg/mL. In still other aspects ofthis embodiment, a hydrogel material comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan is presentat a concentration of, e.g., about 1 mg/mL to about 60 mg/mL, about 10mg/mL to about 40 mg/mL, about 7.5 mg/mL to about 19.5 mg/mL, about 8.5mg/mL to about 18.5 mg/mL, about 9.5 mg/mL to about 17.5 mg/mL, about10.5 mg/mL to about 16.5 mg/mL, about 11.5 mg/mL to about 15.5 mg/mL, orabout 12.5 mg/mL to about 14.5 mg/mL.

In an embodiment, a hydrogel material comprises uncrosslinked hyaluronanpolymers of low molecular weight. In aspects of this embodiment, ahydrogel material comprises a uncrosslinked hyaluronan having a meanmolecular weight of, e.g., about 100,000 Da, about 200,000 Da, about300,000 Da, about 400,000 Da, about 500,000 Da, about 600,000 Da, about700,000 Da, about 800,000 Da, or about 900,000 Da. In yet other aspectsof this embodiment, a hydrogel material comprises uncrosslinkedhyaluronan polymers having a mean molecular weight of, e.g., at most100,000 Da, at most 200,000 Da, at most 300,000 Da, at most 400,000 Da,at most 500,000 Da, at most 600,000 Da, at most 700,000 Da, at most800,000 Da, at most 900,000 Da, or at most 950,000. In still otheraspects of this embodiment, a hydrogel material comprises uncrosslinkedhyaluronan polymers having a mean molecular weight of, e.g., about100,000 Da to about 500,000 Da, about 200,000 Da to about 500,000 Da,about 300,000 Da to about 500,000 Da, about 400,000 Da to about 500,000Da, about 500,000 Da to about 950,000 Da, about 600,000 Da to about950,000 Da, about 700,000 Da to about 950,000 Da, about 800,000 Da toabout 950,000 Da, about 300,000 Da to about 600,000 Da, about 300,000 Dato about 700,000 Da, about 300,000 Da to about 800,000 Da, or about400,000 Da to about 700,000 Da.

In another embodiment, a hydrogel material comprises uncrosslinkedhyaluronan polymers of high molecular weight. In aspects of thisembodiment, a hydrogel material comprises an uncrosslinked hyaluronanhaving a mean molecular weight of, e.g., about 1,000,000 Da, about1,500,000 Da, about 2,000,000 Da, about 2,500,000 Da, about 3,000,000Da, about 3,500,000 Da, about 4,000,000 Da, about 4,500,000 Da, or about5,000,000 Da. In other aspects of this embodiment, a hydrogel materialcomprises an uncrosslinked hyaluronan polymers having a mean molecularweight of, e.g., at least 1,000,000 Da, at least 1,500,000 Da, at least2,000,000 Da, at least 2,500,000 Da, at least 3,000,000 Da, at least3,500,000 Da, at least 4,000,000 Da, at least 4,500,000 Da, or at least5,000,000 Da. In yet other aspects of this embodiment, a hydrogelmaterial comprises an uncrosslinked hyaluronan polymers having a meanmolecular weight of, e.g., about 1,000,000 Da to about 5,000,000 Da,about 1,500,000 Da to about 5,000,000 Da, about 2,000,000 Da to about5,000,000 Da, about 2,500,000 Da to about 5,000,000 Da, about 2,000,000Da to about 3,000,000 Da, about 2,500,000 Da to about 3,500,000 Da, orabout 2,000,000 Da to about 4,000,000 Da. In still other aspects, ahydrogel material comprises an uncrosslinked hyaluronan polymers havinga mean molecular weight of, e.g., greater than 2,000,000 Da and lessthan about 3,000,000 Da, greater than 2,000,000 Da and less than about3,500,000 Da, greater than 2,000,000 Da and less than about 4,000,000Da, greater than 2,000,000 Da and less than about 4,500,000 Da, greaterthan 2,000,000 Da and less than about 5,000,000 Da.

In another embodiment, a hydrogel material comprises uncrosslinkedhyaluronan polymers where the uncrosslinked hyaluronan comprises acombination of both high molecular weight hyaluronan polymers and lowmolecular weight hyaluronan polymers, in various ratios. In aspects ofthis embodiment, a hydrogel material comprises an uncrosslinkedhyaluronan polymers where the uncrosslinked hyaluronan polymerscomprises a combination of both high molecular weight hyaluronanpolymers and low molecular weight hyaluronan polymers in a ratio ofabout 20:1, about 15:1, about 10:1, about 5:1, about 1:1, about 1:5about 1:10, about 1:15, or about 1:20.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising a substantially uncrosslinked glycosaminoglycanpolymer. As sued herein, the term “substantially uncrosslinked” refersto the presence of uncrosslinked glycosaminoglycan polymers in ahydrogel material disclosed herein at a level of at least 90% by weightof the hydrogel material, with the remaining at most 10% by weight ofthe hydrogel material being comprised of other components includingcrosslinked glycosaminoglycan polymers. In aspects of this embodiment, ahydrogel material comprises a substantially uncrosslinked chondroitinsulfate polymer, a substantially uncrosslinked dermatan sulfate polymer,a substantially uncrosslinked keratan sulfate polymer, a substantiallyuncrosslinked heparan polymer, a substantially uncrosslinked heparansulfate polymer, or a substantially uncrosslinked hyaluronan polymer. Inother aspects of this embodiment, a hydrogel material comprises anuncrosslinked glycosaminoglycan where the uncrosslinkedglycosaminoglycan represents, e.g., about 90% or more by weight, about91% or more by weight, about 92% or more by weight, about 93% or more byweight, about 94% or more by weight, about 95% or more by weight, about96% or more by weight, about 97% or more by weight, about 98% or more byweight, or about 99% or more, or about 100% by weight, of the totalglycosaminoglycan present in the hydrogel material. In yet other aspectsof this embodiment, a hydrogel material comprises an uncrosslinkedglycosaminoglycan where the uncrosslinked glycosaminoglycan represents,e.g., about 90% to about 100% by weight, about 93% to about 100% byweight, about 95% to about 100% by weight, or about 97% to about 100% byweight, of the total glycosaminoglycan present in the hydrogel material.

Aspects of the present specification provide, in part, a hydrogelmaterial that is essentially free of a crosslinked glycosaminoglycanpolymer. As used herein, the term “essentially free” (or “consistingessentially of”) refers to a hydrogel material where only trace amountsof cross-linked matrix polymers can be detected. In an aspect of thisembodiment, a hydrogel material comprises a chondroitin sulfate that isessentially free of a crosslinked chondroitin sulfate polymer, adermatan sulfate essentially free of a crosslinked dermatan sulfatepolymer, a keratan sulfate essentially free of a crosslinked keratansulfate polymer, a heparan essentially free of a crosslinked heparanpolymer, a heparan sulfate essentially free of a crosslinked heparansulfate polymer, or a hyaluronan sulfate essentially free of acrosslinked hyaluronan polymer.

Aspects of the present specification provide, in part, a hydrogelmaterial that is entirely free of a crosslinked glycosaminoglycanpolymer. As used herein, the term “entirely free” refers to a hydrogelmaterial that within the detection range of the instrument or processbeing used, crosslinked glycosaminoglycan polymers cannot be detected orits presence cannot be confirmed. In an aspect of this embodiment, ahydrogel material comprises a chondroitin sulfate that is entirely freeof a crosslinked chondroitin sulfate polymer, a dermatan sulfateentirely free of a crosslinked dermatan sulfate polymer, a keratansulfate entirely free of a crosslinked keratan sulfate polymer, aheparan entirely free of a crosslinked heparan polymer, a heparansulfate entirely free of a crosslinked heparan sulfate polymer, or ahyaluronan sulfate entirely free of a crosslinked hyaluronan polymer.

Aspects of the present specification provide, in part, a hydrogelmaterial comprising a ratio of crosslinked glycosaminoglycan polymer anduncrosslinked glycosaminoglycan polymer. This ratio of crosslinked anduncrosslinked glycosaminoglycan polymer is also known as the gel:fluidratio. Any gel:fluid ratio is useful in making the hydrogel materialsdisclosed herein with the proviso that such ratio produces a hydrogelmaterial disclosed herein that improves a skin condition as disclosedherein. Non-limiting examples of gel:fluid ratios include 100:0, 98:2,90:10, 75:25, 70:30, 60:40, 50:50, 40:60, 30:70, 25:75, 10:90; 2:98, and0:100.

In aspects of this embodiment, a hydrogel material comprises acrosslinked glycosaminoglycan polymer and an uncrosslinkedglycosaminoglycan polymer where the gel:fluid ratio is, e.g., about0:100, about 1:99, about 2:98, about 3:97, about 4:96, about 5:95, about6:94, about 7:93, about 8:92, about 9:91, or about 10:90. In otheraspects of this embodiment, a hydrogel material comprises a crosslinkedglycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymerwhere the gel:fluid ratio is, e.g., at most 1:99, at most 2:98, at most3:97, at most 4:96, at most 5:95, at most 6:94, at most 7:93, at most8:92, at most 9:91, or at most 10:90. In yet other aspects of thisembodiment, a hydrogel material comprises a crosslinkedglycosaminoglycan polymer and an uncrosslinked glycosaminoglycan polymerwhere the gel:fluid ratio is, e.g., about 0:100 to about 3:97, about0:100 to about 5:95, or about 0:100 to about 10:90.

In other aspects of this embodiment, a hydrogel material comprises acrosslinked glycosaminoglycan polymer and an uncrosslinkedglycosaminoglycan polymer where the gel:fluid ratio is, e.g., about15:85, about 20:80, about 25:75, about 30:70, about 35:65, about 40:60,about 45:55, about 50:50, about 55:45, about 60:40, about 65:35, about70:30, about 75:25, about 80:20, about 85:15, about 90:10, about 95:5,about 98:2, or about 100:0. In yet other aspects of this embodiment, ahydrogel material comprises a crosslinked glycosaminoglycan polymer andan uncrosslinked glycosaminoglycan polymer where the gel:fluid ratio is,e.g., at most 15:85, at most 20:80, at most 25:75, at most 30:70, atmost 35:65, at most 40:60, at most 45:55, at most 50:50, at most 55:45,at most 60:40, at most 65:35, at most 70:30, at most 75:25, at most80:20, at most 85:15, at most 90:10, at most 95:5, at most 98:2, or atmost 100:0. In still other aspects of this embodiment, a hydrogelmaterial comprises a crosslinked glycosaminoglycan polymer and anuncrosslinked glycosaminoglycan polymer where the gel:fluid ratio is,e.g., about 10:90 to about 70:30, about 15:85 to about 70:30, about10:90 to about 55:45, about 80:20 to about 95:5, about 90:10 to about100:0, about 75:25 to about 100:0, or about 60:40 to about 100:0.

A hydrogel material disclosed herein may be formed into a porousmaterial like a sponge. The porous material can serve both as a bulkingagent to fill the tissue space as well as a substrate to support tissuegrowth. A hydrogel material disclosed herein may be processed into aporous material using standard methods known in the art, including,e.g., freeze-drying, gas foaming, and/or negative templating usingporogens. The pore size of a porous material can be controlled, e.g., byadjusting the concentration of GAG and the particle size of the porogen.After formation of the porous material, the excess porogen is thenextracted, for example, by immersing in water or other suitable solvent.The resultant porous material can then be dried and used. Such methodsare described in, e.g., Kaplin, et al., Concentrated Aqueous SilkFibroin Solution and Use Thereof, U.S. Pat. No. 7,635,755; Ratner andMarshall, Novel Porous Materials, U.S. Patent Publication 2008/0075752;Ma and Chen, Porous Materials having Multi-Sized Geometries, U.S. PatentPublication 2007/0036844; Ma, Reverse Fabrication of Porous Materials,U.S. Patent Publication 2002/0005600; Liu, et al., Porous Materials,Methods of Making and Uses, U.S. Patent Application 61/333,613; and Liu,et al., Porous Materials, Methods of Making and Uses, U.S. PatentApplication 61/333,120, LeBreton et al., Dermal Fillers Comprising SilkFibroin Hydrogels Uses Thereof, U.S. patent application Ser. No.12/883,139; and, Guang-Liang et al., Drug Delivery Platforms ComprisingSilk Fibroin Hydrogels and Uses Thereof, U.S. patent application Ser.No. 12/873,563, each of which is hereby incorporated by reference in itsentirety.

A hydrogel material disclosed herein may be formed into solid particles.The hydrogel particles can serve both as a bulking agent to fill thetissue space as well as a substrate to support tissue growth. A hydrogelmaterial is processed into particles simply by manually or mechanicallypulverizing the material and optionally mixed with a carrier phase suchas, e.g., water or a saline solution to form an injectable or topicalsubstance like a solution, oil, lotion, gel, ointment, cream, slurry,salve, or paste. As such, the disclosed hydrogel materials may bemonophasic or multiphasic compositions. A hydrogel may be milled to aparticle size from about 10 μm to about 1000 μm in diameter. In aspectsof this embodiment, a particle size may be about 15 μm to about 30 μm,about 25 μm to about 50 μm, about 45 μm to about 75 μm, about 70 μm toabout 100 μm, about 50 μm to about 200 μm, about 150 μm to about 300 μm,about 250 μm to about 400 μm, about 350 μm to about 500 μm, about 450 μmto about 600 μm, about 550 μm to about 700 μm, about 650 μm to about 800μm, about 750 μm to about 900 μm, or about 850 μm to about 1000 μm.Saline is then added as a carrier phase by first determining the bulkvolume of a hydrogel material, then vigorously pulverizing the hydrogelinto particles while incorporating an appropriate volume of saline toachieve a desired carrier to hydrogel particle ratio. For example,hydrogel milling may be accomplished by means of a forced sieving ofbulk hydrogel through a series of stainless steel cloth sieves ofdecreasing pore sizes.

Aspects of the present specification provide, in part, a hydrogelmaterial disclosed herein that exhibits a complex modulus, an elasticmodulus, a viscous modulus and/or a tan δ. The compositions as disclosedherein are viscoelastic in that the composition has an elastic component(solid-like such as, e.g., crosslinked glycosaminoglycan polymers) and aviscous component (liquid-like such as, e.g., uncrosslinkedglycosaminoglycan polymers or a carrier phase) when a force is applied(stress, deformation). The rheological attribute that described thisproperty is the complex modulus (G*), which defines a composition'stotal resistance to deformation. The complex modulus is a complex numberwith a real and imaginary part: G*=G′+iG″. The absolute value of G* isAbs(G*)=Sqrt(G′²+G″²). The complex modulus can be defined as the sum ofthe elastic modulus (G′) and the viscous modulus (G″). Falcone, et al.,Temporary Polysaccharide Dermal Fillers: A Model for Persistence Basedon Physical Properties, Dermatol Surg. 35(8): 1238-1243 (2009); Tezel,supra, 2008; Kablik, supra, 2009; Beasley, supra, 2009; each of which ishereby incorporated by reference in its entirety.

Elastic modulus, or modulus of elasticity, refers to the ability of ahydrogel material to resists deformation, or, conversely, an object'stendency to be non-permanently deformed when a force is applied to it.Elastic modulus characterizes the firmness of a composition and is alsoknown as the storage modulus because it describes the storage of energyfrom the motion of the composition. The elastic modulus describes theinteraction between elasticity and strength (G′=stress/strain) and, assuch, provides a quantitative measurement of a composition's hardness orsoftness. The elastic modulus of an object is defined as the slope ofits stress-strain curve in the elastic deformation region:λ=stress/strain, where λ is the elastic modulus in Pascal's; stress isthe force causing the deformation divided by the area to which the forceis applied; and strain is the ratio of the change caused by the stressto the original state of the object. Although depending on the speed atwhich the force is applied, a stiffer composition will have a higherelastic modulus and it will take a greater force to deform the materiala given distance, such as, e.g., an injection. Specifying how stressesare to be measured, including directions, allows for many types ofelastic moduli to be defined. The three primary elastic moduli aretensile modulus, shear modulus, and bulk modulus.

Viscous modulus is also known as the loss modulus because it describesthe energy that is lost as viscous dissipation. Tan δ is the ratio ofthe viscous modulus and the elastic modulus, tan δ=G″/G′. Falcone,supra, 2009. For tan δ values disclosed in the present specification, atan δ is obtained from the dynamic modulus at a frequency of 0.628rad/s. A lower tan δ corresponds to a stiffer, harder, or more elasticcomposition.

Thus, in an embodiment, a hydrogel material disclosed herein exhibits acomplex modulus. In aspects of this embodiment, a hydrogel materialexhibits a complex modulus of, e.g., about 25 Pa, about 50 Pa, about 75Pa, about 100 Pa, about 125 Pa, about 150 Pa, about 175 Pa, about 200Pa, about 250 Pa, about 300 Pa, about 350 Pa, about 400 Pa, about 450Pa, about 500 Pa, about 550 Pa, about 600 Pa, about 650 Pa, about 700Pa, about 750 Pa, or about 800 Pa. In other aspects of this embodiment,a hydrogel material exhibits a complex modulus of, e.g., at most 25 Pa,at most 50 Pa, at most 75 Pa, at most 100 Pa, at most 125 Pa, at most150 Pa, at most 175 Pa, at most 200 Pa, at most 250 Pa, at most 300 Pa,at most 350 Pa, at most 400 Pa, at most 450 Pa, at most 500 Pa, at most550 Pa, at most 600 Pa, at most 650 Pa, at most 700 Pa, at most 750 Pa,or at most 800 Pa. In yet other aspects of this embodiment, a hydrogelmaterial exhibits a complex modulus of, e.g., about 25 Pa to about 150Pa, about 25 Pa to about 300 Pa, about 25 Pa to about 500 Pa, about 25Pa to about 800 Pa, about 125 Pa to about 300 Pa, about 125 Pa to about500 Pa, or about 125 Pa to about 800 Pa.

In another embodiment, a hydrogel material disclosed herein exhibits anelastic modulus. In aspects of this embodiment, a hydrogel materialexhibits an elastic modulus of, e.g., about 25 Pa, about 50 Pa, about 75Pa, about 100 Pa, about 125 Pa, about 150 Pa, about 175 Pa, about 200Pa, about 250 Pa, about 300 Pa, about 350 Pa, about 400 Pa, about 450Pa, about 500 Pa, about 550 Pa, about 600 Pa, about 650 Pa, about 700Pa, about 750 Pa, about 800 Pa, about 850 Pa, about 900 Pa, about 950Pa, about 1,000 Pa, about 1,200 Pa, about 1,300 Pa, about 1,400 Pa,about 1,500 Pa, about 1,600 Pa, about 1700 Pa, about 1800 Pa, about 1900Pa, about 2,000 Pa, about 2,100 Pa, about 2,200 Pa, about 2,300 Pa,about 2,400 Pa, or about 2,500 Pa. In other aspects of this embodiment,a hydrogel material exhibits an elastic modulus of, e.g., at least 25Pa, at least 50 Pa, at least 75 Pa, at least 100 Pa, at least 125 Pa, atleast 150 Pa, at least 175 Pa, at least 200 Pa, at least 250 Pa, atleast 300 Pa, at least 350 Pa, at least 400 Pa, at least 450 Pa, atleast 500 Pa, at least 550 Pa, at least 600 Pa, at least 650 Pa, atleast 700 Pa, at least 750 Pa, at least 800 Pa, at least 850 Pa, atleast 900 Pa, at least 950 Pa, at least 1,000 Pa, at least 1,200 Pa, atleast 1,300 Pa, at least 1,400 Pa, at least 1,500 Pa, at least 1,600 Pa,at least 1700 Pa, at least 1800 Pa, at least 1900 Pa, at least 2,000 Pa,at least 2,100 Pa, at least 2,200 Pa, at least 2,300 Pa, at least 2,400Pa, or at least 2,500 Pa. In yet other aspects of this embodiment, ahydrogel material exhibits an elastic modulus of, e.g., at most 25 Pa,at most 50 Pa, at most 75 Pa, at most 100 Pa, at most 125 Pa, at most150 Pa, at most 175 Pa, at most 200 Pa, at most 250 Pa, at most 300 Pa,at most 350 Pa, at most 400 Pa, at most 450 Pa, at most 500 Pa, at most550 Pa, at most 600 Pa, at most 650 Pa, at most 700 Pa, at most 750 Pa,at most 800 Pa, at most 850 Pa, at most 900 Pa, at most 950 Pa, at most1,000 Pa, at most 1,200 Pa, at most 1,300 Pa, at most 1,400 Pa, at most1,500 Pa, or at most 1,600 Pa. In still other aspects of thisembodiment, a hydrogel material exhibits an elastic modulus of, e.g.,about 25 Pa to about 150 Pa, about 25 Pa to about 300 Pa, about 25 Pa toabout 500 Pa, about 25 Pa to about 800 Pa, about 125 Pa to about 300 Pa,about 125 Pa to about 500 Pa, about 125 Pa to about 800 Pa, about 500 Pato about 1,600 Pa, about 600 Pa to about 1,600 Pa, about 700 Pa to about1,600 Pa, about 800 Pa to about 1,600 Pa, about 900 Pa to about 1,600Pa, about 1,000 Pa to about 1,600 Pa, about 1,100 Pa to about 1,600 Pa,about 1,200 Pa to about 1,600 Pa, about 500 Pa to about 2,500 Pa, about1,000 Pa to about 2,500 Pa, about 1,500 Pa to about 2,500 Pa, about2,000 Pa to about 2,500 Pa, about 1,300 Pa to about 1,600 Pa, about1,400 Pa to about 1,700 Pa, about 1,500 Pa to about 1,800 Pa, about1,600 Pa to about 1,900 Pa, about 1,700 Pa to about 2,000 Pa, about1,800 Pa to about 2,100 Pa, about 1,900 Pa to about 2,200 Pa, about2,000 Pa to about 2,300 Pa, about 2,100 Pa to about 2,400 Pa, or about2,200 Pa to about 2,500 Pa.

In another embodiment, a hydrogel material disclosed herein exhibits atensile modulus. In aspects of this embodiment, a hydrogel materialexhibits a tensile modulus of, e.g., about 1 MPa, about 10 MPa, about 20MPa, about 30 MPa, about 40 MPa, about 50 MPa, about 60 MPa, about 70MPa, about 80 MPa, about 90 MPa, about 100 MPa, about 200 MPa, about 300MPa, about 400 MPa, about 500 MPa, about 750 MPa, about 1 GPa, about 5GPa, about 10 GPa, about 15 GPa, about 20 GPa, about 25 GPa, or about 30GPa. In other aspects of this embodiment, a hydrogel material exhibits atensile modulus of, e.g., at least 1 MPa, at least 10 MPa, at least 20MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa,at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, atleast 200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa, atleast 750 MPa, at least 1 GPa, at least 5 GPa, at least 10 GPa, at least15 GPa, at least 20 GPa, at least 25 GPa, or at least 30 GPa In yetother aspects of this embodiment, a hydrogel material exhibits a tensilemodulus of, e.g., about 1 MPa to about 30 MPa, about 10 MPa to about 50MPa, about 25 MPa to about 75 MPa, about 50 MPa to about 100 MPa, about100 MPa to about 300 MPa, about 200 MPa to about 400 MPa, about 300 MPato about 500 MPa, about 100 MPa to about 500 MPa, about 250 MPa to about750 MPa, about 500 MPa to about 1 GPa, about 1 GPa to about 30 GPa,about 10 GPa to about 30 GPa.

In another embodiment, a hydrogel material disclosed herein exhibitsshear modulus. In aspects of this embodiment, a hydrogel materialexhibits a shear modulus of, e.g., about 1 MPa, about 10 MPa, about 20MPa, about 30 MPa, about 40 MPa, about 50 MPa, about 60 MPa, about 70MPa, about 80 MPa, about 90 MPa, about 100 MPa, about 200 MPa, about 300MPa, about 400 MPa, about 500 MPa, about 750 MPa, about 1 GPa, about 5GPa, about 10 GPa, about 15 GPa, about 20 GPa, about 25 GPa, or about 30GPa. In other aspects of this embodiment, a hydrogel material exhibits ashear modulus of, e.g., at least 1 MPa, at least 10 MPa, at least 20MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60 MPa,at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100 MPa, atleast 200 MPa, at least 300 MPa, at least 400 MPa, at least 500 MPa, atleast 750 MPa, at least 1 GPa, at least 5 GPa, at least 10 GPa, at least15 GPa, at least 20 GPa, at least 25 GPa, or at least 30 GPa In yetother aspects of this embodiment, a hydrogel material exhibits a shearmodulus of, e.g., about 1 MPa to about 30 MPa, about 10 MPa to about 50MPa, about 25 MPa to about 75 MPa, about 50 MPa to about 100 MPa, about100 MPa to about 300 MPa, about 200 MPa to about 400 MPa, about 300 MPato about 500 MPa, about 100 MPa to about 500 MPa, about 250 MPa to about750 MPa, about 500 MPa to about 1 GPa, about 1 GPa to about 30 GPa,about 10 GPa to about 30 GPa.

In another embodiment, a hydrogel material disclosed herein exhibits abulk modulus. In aspects of this embodiment, a hydrogel materialexhibits a bulk modulus of, e.g., about 5 GPa, about 6 GPa, about 7 GPa,about 8 GPa, about 9 GPa, about 10 GPa, about 15 GPa, about 20 GPa,about 25 GPa, about 30 GPa, about 35 GPa, about 40 GPa, about 45 GPa,about 50 GPa, about 60 GPa, about 70 GPa, about 80 GPa, about 90 GPa,about 100 GPa. In other aspects of this embodiment, a hydrogel materialexhibits a bulk modulus of, e.g., at least 5 GPa, at least 6 GPa, atleast 7 GPa, at least 8 GPa, at least 9 GPa, at least 10 GPa, at least15 GPa, at least 20 GPa, at least 25 GPa, at least 30 GPa, at least 35GPa, at least 40 GPa, at least 45 GPa, at least 50 GPa, at least 60 GPa,at least 70 GPa, at least 80 GPa, at least 90 GPa, at least 100 GPa. Inyet other aspects of this embodiment, a hydrogel material exhibits abulk modulus of, e.g., about 5 GPa to about 50 GPa, about 5 GPa to about100 GPa, about 10 GPa to about 50 GPa, about 10 GPa to about 100 GPa, orabout 50 GPa to about 100 GPa.

In another embodiment, a hydrogel material disclosed herein exhibits aviscous modulus. In aspects of this embodiment, a hydrogel materialexhibits a viscous modulus of, e.g., about 10 Pa, about 20 Pa, about 30Pa, about 40 Pa, about 50 Pa, about 60 Pa, about 70 Pa, about 80 Pa,about 90 Pa, about 100 Pa, about 150 Pa, about 200 Pa, about 250 Pa,about 300 Pa, about 350 Pa, about 400 Pa, about 450 Pa, about 500 Pa,about 550 Pa, about 600 Pa, about 650 Pa, or about 700 Pa. In otheraspects of this embodiment, a hydrogel material exhibits a viscousmodulus of, e.g., at most 10 Pa, at most 20 Pa, at most 30 Pa, at most40 Pa, at most 50 Pa, at most 60 Pa, at most 70 Pa, at most 80 Pa, atmost 90 Pa, at most 100 Pa, at most 150 Pa, at most 200 Pa, at most 250Pa, at most 300 Pa, at most 350 Pa, at most 400 Pa, at most 450 Pa, atmost 500 Pa, at most 550 Pa, at most 600 Pa, at most 650 Pa, or at most700 Pa. In yet other aspects of this embodiment, a hydrogel materialexhibits a viscous modulus of, e.g., about 10 Pa to about 30 Pa, about10 Pa to about 50 Pa, about 10 Pa to about 100 Pa, about 10 Pa to about150 Pa, about 70 Pa to about 100 Pa, about 50 Pa to about 350 Pa, about150 Pa to about 450 Pa, about 250 Pa to about 550 Pa, about 350 Pa toabout 700 Pa, about 50 Pa to about 150 Pa, about 100 Pa to about 200 Pa,about 150 Pa to about 250 Pa, about 200 Pa to about 300 Pa, about 250 Pato about 350 Pa, about 300 Pa to about 400 Pa, about 350 Pa to about 450Pa, about 400 Pa to about 500 Pa, about 450 Pa to about 550 Pa, about500 Pa to about 600 Pa, about 550 Pa to about 650 Pa, or about 600 Pa toabout 700 Pa.

In another embodiment, a hydrogel material disclosed herein exhibits atan δ. In aspects of this embodiment, a hydrogel material exhibits a tanδ of, e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2,about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8, about1.9, about 2.0, about 2.1, about 2.2, about 2.3, about 2.4, or about2.5. In other aspects of this embodiment, a hydrogel material exhibits atan δ of, e.g., at most 0.1, at most 0.2, at most 0.3, at most 0.4, atmost 0.5, at most 0.6, at most 0.7, at most 0.8, at most 0.9, at most1.0, at most 1.1, at most 1.2, at most 1.3, at most 1.4, at most 1.5, atmost 1.6, at most 1.7, at most 1.8, at most 1.9, at most 2.0, at most2.1, at most 2.2, at most 2.3, at most 2.4, or at most 2.5. In yet otheraspects of this embodiment, a hydrogel material exhibits a tan δ of,e.g., about 0.1 to about 0.3, about 0.3 to about 0.5, about 0.5 to about0.8, about 1.1 to about 1.4, about 1.4 to about 1.7, about 0.3 to about0.6, about 0.1 to about 0.5, about 0.5 to about 0.9, about 0.1 to about0.6, about 0.1 to about 1.0, about 0.5 to about 1.5, about 1.0 to about2.0, or about 1.5 to about 2.5.

Aspects of the present specification provide, in part, a hydrogelmaterial disclosed herein having hardness. Hardness refers to variousproperties of an object in the solid phase that gives it high resistanceto various kinds of shape change when force is applied. Hardness ismeasured using a durometer and is a unitless value that ranges from zeroto 100. The ability or inability of a hydrogel to be easily compressedwill affect its suitability for application in different tissuereplacement roles, i.e., mechanical compliance as bone, fat, connectivetissue. Hardness will also affect the ability of a hydrogel to beeffectively comminuted, the reason being that a hard material may bemore easily and consistently comminuted. Hardness will also affectextrudability, as a soft material may be more readily able to beslightly compressed during injection to pack with other particles orchange shape to pass through a syringe barrel or needle.

In an embodiment, a hydrogel material disclosed herein exhibits lowhardness. In aspects of this embodiment, a hydrogel material exhibits ahardness of, e.g., about 5, about 10, about 15, about 20, about 25,about 30, or about 35. In other aspects of this embodiment, a hydrogelmaterial exhibits a hardness of, e.g., at most 5, at most 10, at most15, at most 20, at most 25, at most 30, or at most 35. In yet otheraspects of this embodiment, a hydrogel material exhibits a hardness of,e.g., about 5 to about 35, about 10 to about 35, about 15 to about 35,about 20 to about 35, or about 25 to about 35, about 5 to about 40,about 10 to about 40, about 15 to about 40, about 20 to about 40, about25 to about 40, or about 30 to about 40.

In an embodiment, a hydrogel material disclosed herein exhibits mediumhardness. In aspects of this embodiment, a hydrogel material exhibits ahardness of, e.g., about 40, about 45, about 50, about 55, or about 60.In other aspects of this embodiment, a hydrogel material exhibits ahardness of, e.g., at least 40, at least 45, at least 50, at least 55,or at least 60. In yet other aspects of this embodiment, a hydrogelmaterial exhibits a hardness of, e.g., at most 40, at most 45, at most50, at most 55, or at most 60. In still other aspects of thisembodiment, a hydrogel material exhibits a hardness of, e.g., about 35to about 60, about 35 to about 55, about 35 to about 50, about 35 toabout 45, about 40 to about 60, about 45 to about 60, about 50 to about60, about 55 to about 60, about 40 to about 65, about 45 to about 65,about 50 to about 65, about 55 to about 65.

In another embodiment, a hydrogel material disclosed herein exhibitshigh hardness. In aspects of this embodiment, a hydrogel materialexhibits a hardness of, e.g., about 65, about 70, about 75, about 80,about 85, about 90, about 95, or about 100. In other aspects of thisembodiment, a hydrogel material exhibits a hardness of, e.g., at least65, at least 70, at least 75, at least 80, at least 85, at least 90, atleast 95, or at least 100. In yet other aspects of this embodiment, ahydrogel material exhibits a hardness of, e.g., about 65 to about 100,about 70 to about 100, about 75 to about 100, about 80 to about 100,about 85 to about 100, about 90 to about 100, about 65 to about 75,about 65 to about 80, about 65 to about 85, about 65 to about 90, about65 to about 95, about 60 to about 75, about 60 to about 80, about 60 toabout 85, about 60 to about 90, or about 60 to about 95.

In an embodiment, a hydrogel material disclosed herein exhibits highresistant to deformation. In aspects of this embodiment, a hydrogelmaterial exhibits resistant to deformation of, e.g., about 100%, about99%, about 98%, about 97%, about 96%, about 95%, about 94%, about 93%,about 92%, about 91%, about 90%, about 89%, about 88%, about 87%, about86%, or about 85%. In other aspects of this embodiment, a hydrogelmaterial exhibits resistant to deformation of, e.g., at least 99%, atleast 98%, at least 97%, at least 96%, at least 95%, at least 94%, atleast 93%, at least 92%, at least 91%, at least 90%, at least 89%, atleast 88%, at least 87%, at least 86%, or at least 85%. In yet otheraspects of this embodiment, a hydrogel material exhibits resistant todeformation of, e.g., at most 99%, at most 98%, at most 97%, at most96%, at most 95%, at most 94%, at most 93%, at most 92%, at most 91%, atmost 90%, at most 89%, at most 88%, at most 87%, at most 86%, or at most85%. In still aspects of this embodiment, a hydrogel material exhibitsresistant to deformation of, e.g., about 85% to about 100%, about 87% toabout 100%, about 90% to about 100%, about 93% to about 100%, about 95%to about 100%, or about 97% to about 100%.

A hydrogel material disclosed herein exhibits high tensile strength.Tensile strength has three different definitional points of stressmaxima. Yield strength refers to the stress at which material strainchanges from elastic deformation to plastic deformation, causing it todeform permanently. Ultimate strength refers to the maximum stress amaterial can withstand when subjected to tension, compression orshearing. It is the maximum stress on the stress-strain curve. Breakingstrength refers to the stress coordinate on the stress-strain curve atthe point of rupture, or when the material pulls apart.

In another embodiment, a hydrogel material disclosed herein exhibitshigh yield strength relative to other polymer classes. In aspects ofthis embodiment, a silk fibroin material exhibits a yield strength of,e.g., about 0.1 MPa, about 0.5 MPa, about 1 MPa, about 5 MPa, about 10MPa, about 20 MPa, about 30 MPa, about 40 MPa, about 50 MPa, about 60MPa, about 70 MPa, about 80 MPa, about 90 MPa, about 100 MPa, about 200MPa, about 300 MPa, about 400 MPa, about 500 MPa. In other aspects ofthis embodiment, a hydrogel material exhibits a yield strength of, e.g.,at least 0.1 MPa, at least 0.5 MPa, at least 1 MPa, at least 5 MPa, atleast 10 MPa, at least 20 MPa, at least 30 MPa, at least 40 MPa, atleast 50 MPa, at least 60 MPa, at least 70 MPa, at least 80 MPa, atleast 90 MPa, at least 100 MPa, at least 200 MPa, at least 300 MPa, atleast 400 MPa, at least 500 MPa. In yet other aspects of thisembodiment, a hydrogel material exhibits a yield strength of, e.g., atmost 1 MPa, at most 5 MPa, at most 10 MPa, at most 20 MPa, at most 30MPa, at most 40 MPa, at most 50 MPa, at most 60 MPa, at most 70 MPa, atmost 80 MPa, at most 90 MPa, at most 100 MPa, at most 200 MPa, at most300 MPa, at most 400 MPa, at most 500 MPa, at most 600 MPa, at most 700MPa, at most 800 MPa, at most 900 MPa, at most 1000 MPa, at most 1500MPa, or at most 2000 MPa. In still other aspects of this embodiment, ahydrogel material exhibits a yield strength of, e.g., about 1 MPa toabout 50 MPa, about 1 MPa to about 60 MPa, about 1 MPa to about 70 MPa,about 1 MPa to about 80 MPa, about 1 MPa to about 90 MPa, about 1 MPa toabout 100 MPa, about 10 MPa to about 50 MPa, about 10 MPa to about 60MPa, about 10 MPa to about 70 MPa, about 10 MPa to about 80 MPa, about10 MPa to about 90 MPa, about 10 MPa to about 100 MPa, about 10 MPa toabout 200 MPa, about 10 MPa to about 300 MPa, or about 100 MPa to about300 MPa.

In another embodiment, a hydrogel material disclosed herein exhibitshigh ultimate strength. In aspects of this embodiment, a hydrogelmaterial exhibits an ultimate strength of, e.g., about 0.1 MPa, about0.5 MPa, about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 30MPa, about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80MPa, about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about400 MPa, about 500 MPa. In other aspects of this embodiment, a hydrogelmaterial exhibits an ultimate strength of, e.g., at least 0.1 MPa, atleast 0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, at least20 MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60MPa, at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100MPa, at least 200 MPa, at least 300 MPa, at least 400 MPa, at least 500MPa. In yet other aspects of this embodiment, a hydrogel materialexhibits an ultimate strength of, e.g., at most 1 MPa, at most 5 MPa, atmost 10 MPa, at most 20 MPa, at most 30 MPa, at most 40 MPa, at most 50MPa, at most 60 MPa, at most 70 MPa, at most 80 MPa, at most 90 MPa, atmost 100 MPa, at most 200 MPa, at most 300 MPa, at most 400 MPa, at most500 MPa, at most 600 MPa, at most 700 MPa, at most 800 MPa, at most 900MPa, at most 1000 MPa, at most 1500 MPa, or at most 2000 MPa. In stillother aspects of this embodiment, a hydrogel material exhibits anultimate strength of, e.g., about 1 MPa to about 50 MPa, about 1 MPa toabout 60 MPa, about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa,about 1 MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPato about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about 70MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa, about10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about 10 MPa toabout 300 MPa, or about 100 MPa to about 300 MPa.

In another embodiment, a hydrogel material disclosed herein exhibitshigh breaking strength. In aspects of this embodiment, a hydrogelmaterial exhibits a breaking strength of, e.g., about 0.1 MPa, about 0.5MPa, about 1 MPa, about 5 MPa, about 10 MPa, about 20 MPa, about 30 MPa,about 40 MPa, about 50 MPa, about 60 MPa, about 70 MPa, about 80 MPa,about 90 MPa, about 100 MPa, about 200 MPa, about 300 MPa, about 400MPa, about 500 MPa. In other aspects of this embodiment, a hydrogelmaterial exhibits a breaking strength of, e.g., at least 0.1 MPa, atleast 0.5 MPa, at least 1 MPa, at least 5 MPa, at least 10 MPa, at least20 MPa, at least 30 MPa, at least 40 MPa, at least 50 MPa, at least 60MPa, at least 70 MPa, at least 80 MPa, at least 90 MPa, at least 100MPa, at least 200 MPa, at least 300 MPa, at least 400 MPa, at least 500MPa. In yet other aspects of this embodiment, a hydrogel materialexhibits a breaking strength of, e.g., at most 1 MPa, at most 5 MPa, atmost 10 MPa, at most 20 MPa, at most 30 MPa, at most 40 MPa, at most 50MPa, at most 60 MPa, at most 70 MPa, at most 80 MPa, at most 90 MPa, atmost 100 MPa, at most 200 MPa, at most 300 MPa, at most 400 MPa, at most500 MPa, at most 600 MPa, at most 700 MPa, at most 800 MPa, at most 900MPa, at most 1000 MPa, at most 1500 MPa, or at most 2000 MPa. In stillother aspects of this embodiment, a hydrogel material exhibits abreaking strength of, e.g., about 1 MPa to about 50 MPa, about 1 MPa toabout 60 MPa, about 1 MPa to about 70 MPa, about 1 MPa to about 80 MPa,about 1 MPa to about 90 MPa, about 1 MPa to about 100 MPa, about 10 MPato about 50 MPa, about 10 MPa to about 60 MPa, about 10 MPa to about 70MPa, about 10 MPa to about 80 MPa, about 10 MPa to about 90 MPa, about10 MPa to about 100 MPa, about 10 MPa to about 200 MPa, about 10 MPa toabout 300 MPa, or about 100 MPa to about 300 MPa.

Aspects of the present specification provide, in part, a hydrogelmaterial disclosed herein having a transparency and/or translucency.Transparency (also called pellucidity or diaphaneity) is the physicalproperty of allowing light to pass through a material, whereastranslucency (also called translucence or translucidity) only allowslight to pass through diffusely. The opposite property is opacity.Transparent materials are clear, while translucent ones cannot be seenthrough clearly. The silk fibroin hydrogels disclosed herein may, or maynot, exhibit optical properties such as transparency and translucency.In certain cases, e.g., superficial line filling, it would be anadvantage to have an opaque hydrogel. In other cases such as developmentof a lens or a “humor” for filling the eye, it would be an advantage tohave a translucent hydrogel. These properties could be modified byaffecting the structural distribution of the hydrogel material. Factorsused to control a hydrogel's optical properties include, withoutlimitation, polymer concentration, gel crystallinity, and hydrogelhomogeneity.

When light encounters a material, it can interact with it in severaldifferent ways. These interactions depend on the nature of the light(its wavelength, frequency, energy, etc.) and the nature of thematerial. Light waves interact with an object by some combination ofreflection, and transmittance with refraction. As such, an opticallytransparent material allows much of the light that falls on it to betransmitted, with little light being reflected. Materials which do notallow the transmission of light are called optically opaque or simplyopaque.

In an embodiment, a hydrogel material disclosed herein is opticallytransparent. In aspects of this embodiment, a hydrogel materialtransmits, e.g., about 75% of the light, about 80% of the light, about85% of the light, about 90% of the light, about 95% of the light, orabout 100% of the light. In other aspects of this embodiment, a hydrogelmaterial transmits, e.g., at least 75% of the light, at least 80% of thelight, at least 85% of the light, at least 90% of the light, or at least95% of the light. In yet other aspects of this embodiment, a hydrogelmaterial transmits, e.g., about 75% to about 100% of the light, about80% to about 100% of the light, about 85% to about 100% of the light,about 90% to about 100% of the light, or about 95% to about 100% of thelight.

In another embodiment, a hydrogel material disclosed herein is opticallyopaque. In aspects of this embodiment, a hydrogel material transmits,e.g., about 5% of the light, about 10% of the light, about 15% of thelight, about 20% of the light, about 25% of the light, about 30% of thelight, about 35% of the light, about 40% of the light, about 45% of thelight, about 50% of the light, about 55% of the light, about 60% of thelight, about 65% of the light, or about 70% of the light. In otheraspects of this embodiment, a hydrogel material transmits, e.g., at most5% of the light, at most 10% of the light, at most 15% of the light, atmost 20% of the light, at most 25% of the light, at most 30% of thelight, at most 35% of the light, at most 40% of the light, at most 45%of the light, at most 50% of the light, at most 55% of the light, atmost 60% of the light, at most 65% of the light, at most 70% of thelight, or at most 75% of the light. In other aspects of this embodiment,a hydrogel material transmits, e.g., about 5% to about 15%, about 5% toabout 20%, about 5% to about 25%, about 5% to about 30%, about 5% toabout 35%, about 5% to about 40%, about 5% to about 45%, about 5% toabout 50%, about 5% to about 55%, about 5% to about 60%, about 5% toabout 65%, about 5% to about 70%, about 5% to about 75%, about 15% toabout 20%, about 15% to about 25%, about 15% to about 30%, about 15% toabout 35%, about 15% to about 40%, about 15% to about 45%, about 15% toabout 50%, about 15% to about 55%, about 15% to about 60%, about 15% toabout 65%, about 15% to about 70%, about 15% to about 75%, about 25% toabout 35%, about 25% to about 40%, about 25% to about 45%, about 25% toabout 50%, about 25% to about 55%, about 25% to about 60%, about 25% toabout 65%, about 25% to about 70%, or about 25% to about 75%, of thelight.

In an embodiment, a hydrogel material disclosed herein is opticallytranslucent. In aspects of this embodiment, a hydrogel materialdiffusely transmits, e.g., about 75% of the light, about 80% of thelight, about 85% of the light, about 90% of the light, about 95% of thelight, or about 100% of the light. In other aspects of this embodiment,a hydrogel material diffusely transmits, e.g., at least 75% of thelight, at least 80% of the light, at least 85% of the light, at least90% of the light, or at least 95% of the light. In yet other aspects ofthis embodiment, a hydrogel material diffusely transmits, e.g., about75% to about 100% of the light, about 80% to about 100% of the light,about 85% to about 100% of the light, about 90% to about 100% of thelight, or about 95% to about 100% of the light.

Aspects of the present specification provide, in part, a hydrogelmaterial disclosed herein that exhibits a dynamic viscosity. Viscosityis resistance of a fluid to shear or flow caused by either shear stressor tensile stress. Viscosity describes a fluid's internal resistance toflow caused by intermolecular friction exerted when layers of fluidsattempt to slide by one another and may be thought of as a measure offluid friction. The less viscous the fluid, the greater its ease ofmovement (fluidity).

Viscosity can be defined in two ways; dynamic viscosity (μ, although ηis sometimes used) or kinematic viscosity (v). Dynamic viscosity, alsoknown as absolute or complex viscosity, is the tangential force per unitarea required to move one horizontal plane with respect to the other atunit velocity when maintained a unit distance apart by the fluid. The SIphysical unit of dynamic viscosity is the Pascal-second (Pa·s), which isidentical to N·m−2·s. Dynamic viscosity can be expressed as T=μdvx/dz,where T=shearing stress, μ=dynamic viscosity, and dvx/dz is the velocitygradient over time. For example, if a fluid with a viscosity of one Pa·sis placed between two plates, and one plate is pushed sideways with ashear stress of one Pascal, it moves a distance equal to the thicknessof the layer between the plates in one second. Dynamic viscositysymbolize by is also used, is measured with various types of rheometers,devices used to measure the way in which a liquid, suspension or slurryflows in response to applied forces.

Kinematic viscosity (v) is the ratio of dynamic viscosity to density, aquantity in which no force is involved and is defined as follows: v=μ/ρ,where μ is the dynamic viscosity ρ is density with the SI unit of kg/m³.Kinematic viscosity is usually measured by a glass capillary viscometeras has an SI unit of m²/s.

The viscosity of a material is highly temperature dependent and foreither dynamic or kinematic viscosity to be meaningful, the referencetemperature must be quoted. For the viscosity values disclosed herein, adynamic viscosity is measured at 1 Pa with a cone/plane geometry 2°/40cm and a temperature of 20° C. Examples of the dynamic viscosity ofvarious fluids at 20° C. is as follows: water is about 1.0×10⁻³ Pa·s,blood is about 3-4×10⁻³ Pa·s, vegetable oil is about 60-85×10⁻³ Pa·s,motor oil SE 30 is about 0.2 Pa·s, glycerin is about 1.4 Pa·s, maplesyrup is about 2-3 Pa·s, honey is about 10 Pa·s, chocolate syrup isabout 10-25 Pa·s, peanut butter is about 150-250 Pa·s, lard is about1,000 Pa·s, vegetable shortening is about 1,200 Pa·s, and tar is about30,000 Pa·s.

In aspects of this embodiment, a hydrogel material disclosed hereinexhibits a dynamic viscosity of, e.g., about 10 Pa·s, about 20 Pa·s,about 30 Pa·s, about 40 Pa·s, about 50 Pa·s, about 60 Pa·s, about 70Pa·s, about 80 Pa·s, about 90 Pa·s, about 100 Pa·s, about 125 Pa·s,about 150 Pa·s, about 175 Pa·s, about 200 Pa·s, about 225 Pa·s, about250 Pa·s, about 275 Pa·s, about 300 Pa·s, about 400 Pa·s, about 500Pa·s, about 600 Pa·s, about 700 Pa·s, about 750 Pa·s, about 800 Pa·s,about 900 Pa·s, about 1,000 Pa·s, about 1,100 Pa·s, or about 1,200 Pa·s.In other aspects of this embodiment, a hydrogel material disclosedherein exhibits a dynamic viscosity of, e.g., at most 10 Pa·s, at most20 Pa·s, at most 30 Pa·s, at most 40 Pa·s, at most 50 Pa·s, at most 60Pa·s, at most 70 Pa·s, at most 80 Pa·s, at most 90 Pa·s, at most 100Pa·s, at most 125 Pa·s, at most 150 Pa·s, at most 175 Pa·s, at most 200Pa·s, at most 225 Pa·s, at most 250 Pa·s, at most 275 Pa·s, at most 300Pa·s, at most 400 Pa·s, at most 500 Pa·s, at most 600 Pa·s, at most 700Pa·s, at most 750 Pa·s, at most 800 Pa·s, at most 900 Pa·s, or at most1000 Pa·s. In yet other aspects of this embodiment, a hydrogel materialdisclosed herein exhibits a dynamic viscosity of, e.g., about 10 Pa·s toabout 100 Pa·s, about 10 Pa·s to about 150 Pa·s, about 10 Pa·s to about250 Pa·s, about 50 Pa·s to about 100 Pa·s, about 50 Pa·s to about 150Pa·s, about 50 Pa·s to about 250 Pa·s, about 100 Pa·s to about 500 Pa·s,about 100 Pa·s to about 750 Pa·s, about 100 Pa·s to about 1,000 Pa·s,about 100 Pa·s to about 1,200 Pa·s, about 300 Pa·s to about 500 Pa·s,about 300 Pa·s to about 750 Pa·s, about 300 Pa·s to about 1,000 Pa·s, orabout 300 Pa·s to about 1,200 Pa·s.

Aspects of the present specification provide, in part, a hydrogelmaterial disclosed herein that exhibits cohesivity. Cohesivity, alsoreferred to as cohesion cohesive attraction, cohesive force, orcompression force is a physical property of a material, caused by theintermolecular attraction between like-molecules within the materialthat acts to unite the molecules. Cohesivity is expressed in terms ofgrams-force (gmf). Cohesiveness is affected by, among other factors, themolecular weight ratio of the initial free glycosaminoglycan polymer,the degree of crosslinking of glycosaminoglycan polymers, the amount ofresidual free glycosaminoglycan polymers following crosslinking, and thepH of the hydrogel material. A composition should be sufficientlycohesive as to remain localized to a site of administration.Additionally, in certain applications, a sufficient cohesiveness isimportant for a composition to retain its shape, and thus functionality,in the event of mechanical load cycling. As such, in one embodiment, ahydrogel material disclosed herein exhibits cohesivity, on par withwater. In yet another embodiment, a hydrogel material disclosed hereinexhibits sufficient cohesivity to remain localized to a site ofadministration. In still another embodiment, a hydrogel materialdisclosed herein exhibits sufficient cohesivity to retain its shape. Ina further embodiment, a hydrogel material disclosed herein exhibitssufficient cohesivity to retain its shape and functionality.

In aspects of this embodiment, a hydrogel material disclosed herein hasa cohesivity of, e.g., about 10 gmf, about 20 gmf, about 30 gmf, about40 gmf, about 50 gmf, about 60 gmf, about 70 gmf, about 80 gmf, about 90gmf, about 100 gmf, about 150 gmf, or about 200 gmf. In other aspects ofthis embodiment, a hydrogel material disclosed herein has a cohesivityof, e.g., at least 10 gmf, at least 20 gmf, at least 30 gmf, at least 40gmf, at least 50 gmf, at least 60 gmf, at least 70 gmf, at least 80 gmf,at least 90 gmf, at least 100 gmf, at least 150 gmf, or at least 200gmf. In yet other aspects of this embodiment, a hydrogel materialdisclosed herein has a cohesivity of, e.g., at most 10 gmf, at most 20gmf, at most 30 gmf, at most 40 gmf, at most 50 gmf, at most 60 gmf, atmost 70 gmf, at most 80 gmf, at most 90 gmf, at most 100 gmf, at most150 gmf, or at most 200 gmf. In yet other aspects of this embodiment, ahydrogel material disclosed herein has a cohesivity of, e.g., about 50gmf to about 150 gmf, about 60 gmf to about 140 gmf, about 70 gmf toabout 130 gmf, about 80 gmf to about 120 gmf, or about 90 gmf to about110 gmf.

In yet other aspects of this embodiment, a hydrogel material disclosedherein has a cohesivity of, e.g., about 10 gmf to about 50 gmf, about 25gmf to about 75 gmf, about 50 gmf to about 150 gmf, about 100 gmf toabout 200 gmf, about 100 gmf to about 300 gmf, about 100 gmf to about400 gmf, about 100 gmf to about 500 gmf, about 200 gmf to about 300 gmf,about 200 gmf to about 400 gmf, about 200 gmf to about 500 gmf, about200 gmf to about 600 gmf, about 200 gmf to about 700 gmf, about 300 gmfto about 400 gmf, about 300 gmf to about 500 gmf, about 300 gmf to about600 gmf, about 300 gmf to about 700 gmf, about 300 gmf to about 800 gmf,about 400 gmf to about 500, about 400 gmf to about 600, about 400 gmf toabout 700, about 400 gmf to about 800, about 500 gmf to about 600 gmf,about 500 gmf to about 700 gmf, about 500 gmf to about 800 gmf, about600 gmf to about 700 gmf, about 600 gmf to about 800 gmf, about 700 gmfto about 800 gmf, about 1000 gmf to about 2000 gmf, about 1000 gmf toabout 3000 gmf, or about 2000 gmf to about 3000 gmf.

Aspects of the present specification provide, in part, a hydrogelmaterial disclosed herein that exhibits a physiologically-acceptableosmolarity. As used herein, the term “osmolarity” refers to theconcentration of osmotically active solutes in solution. As used herein,the term “a physiologically-acceptable osmolarity” refers to anosmolarity in accord with, or characteristic of, the normal functioningof a living organism. As such, administration of a hydrogel material asdisclosed herein exhibits an osmolarity that has substantially no longterm or permanent detrimental effect when administered to a mammal.Osmolarity is expressed in terms of osmoles of osmotically active soluteper liter of solvent (Osmol/L or Osm/L). Osmolarity is distinct frommolarity because it measures moles of osmotically active soluteparticles rather than moles of solute. The distinction arises becausesome compounds can dissociate in solution, whereas others cannot. Theosmolarity of a solution can be calculated from the followingexpression: Osmol/L=Σφ_(i)η_(i)C_(i), where φ is the osmoticcoefficient, which accounts for the degree of non-ideality of thesolution; ρ is the number of particles (e.g. ions) into which a moleculedissociates; and C is the molar concentration of the solute; and i isthe index representing the identity of a particular solute. Theosmolarity of a hydrogel material disclosed herein can be measured usinga conventional method that measures solutions.

In an embodiment, a hydrogel material disclosed herein exhibits aphysiologically-acceptable osmolarity. In aspects of this embodiment, ahydrogel material exhibits an osmolarity of, e.g., about 100 mOsm/L,about 150 mOsm/L, about 200 mOsm/L, about 250 mOsm/L, about 300 mOsm/L,about 350 mOsm/L, about 400 mOsm/L, about 450 mOsm/L, or about 500mOsm/L. In other aspects of this embodiment, a hydrogel materialexhibits an osmolarity of, e.g., at least 100 mOsm/L, at least 150mOsm/L, at least 200 mOsm/L, at least 250 mOsm/L, at least 300 mOsm/L,at least 350 mOsm/L, at least 400 mOsm/L, at least 450 mOsm/L, or atleast 500 mOsm/L. In yet other aspects of this embodiment, a hydrogelmaterial exhibits an osmolarity of, e.g., at most 100 mOsm/L, at most150 mOsm/L, at most 200 mOsm/L, at most 250 mOsm/L, at most 300 mOsm/L,at most 350 mOsm/L, at most 400 mOsm/L, at most 450 mOsm/L, or at most500 mOsm/L. In still other aspects of this embodiment, a hydrogelmaterial exhibits an osmolarity of, e.g., about 100 mOsm/L to about 500mOsm/L, about 200 mOsm/L to about 500 mOsm/L, about 200 mOsm/L to about400 mOsm/L, about 300 mOsm/L to about 400 mOsm/L, about 270 mOsm/L toabout 390 mOsm/L, about 225 mOsm/L to about 350 mOsm/L, about 250 mOsm/Lto about 325 mOsm/L, about 275 mOsm/L to about 300 mOsm/L, or about 285mOsm/L to about 290 mOsm/L.

Aspects of the present specification provide, in part, a hydrogelmaterial disclosed herein that exhibits a physiologically-acceptableosmolality. As used herein, the term “osmolality” refers to theconcentration of osmotically active solutes per kilo of solvent in thebody. As used herein, the term “a physiologically-acceptable osmolality”refers to an osmolality in accord with, or characteristic of, the normalfunctioning of a living organism. As such, administration of a hydrogelmaterial disclosed herein exhibits an osmolality that has substantiallyno long term or permanent detrimental effect when administered to amammal. Osmolality is expressed in terms of osmoles of osmoticallyactive solute per kilogram of solvent (osmol/kg or Osm/kg) and is equalto the sum of the molalities of all the solutes present in thatsolution. The osmolality of a solution can be measured using anosmometer. The most commonly used instrument in modern laboratories is afreezing point depression osmometer. This instruments measure the changein freezing point that occurs in a solution with increasing osmolality(freezing point depression osmometer) or the change in vapor pressurethat occurs in a solution with increasing osmolality (vapor pressuredepression osmometer).

In an embodiment, a hydrogel material disclosed herein exhibits aphysiologically-acceptable osmolality. In aspects of this embodiment, ahydrogel material exhibits an osmolality of, e.g., about 100 mOsm/kg,about 150 mOsm/kg, about 200 mOsm/kg, about 250 mOsm/kg, about 300mOsm/kg, about 350 mOsm/kg, about 400 mOsm/kg, about 450 mOsm/kg, orabout 500 mOsm/kg. In other aspects of this embodiment, a hydrogelmaterial exhibits an osmolality of, e.g., at least 100 mOsm/kg, at least150 mOsm/kg, at least 200 mOsm/kg, at least 250 mOsm/kg, at least 300mOsm/kg, at least 350 mOsm/kg, at least 400 mOsm/kg, at least 450mOsm/kg, or at least 500 mOsm/kg. In yet other aspects of thisembodiment, a hydrogel material exhibits an osmolality of, e.g., at most100 mOsm/kg, at most 150 mOsm/kg, at most 200 mOsm/kg, at most 250mOsm/kg, at most 300 mOsm/kg, at most 350 mOsm/kg, at most 400 mOsm/kg,at most 450 mOsm/kg, or at most 500 mOsm/kg. In still other aspects ofthis embodiment, a hydrogel material exhibits an osmolality of, e.g.,about 100 mOsm/kg to about 500 mOsm/kg, about 200 mOsm/kg to about 500mOsm/kg, about 200 mOsm/kg to about 400 mOsm/kg, about 300 mOsm/kg toabout 400 mOsm/kg, about 270 mOsm/kg to about 390 mOsm/kg, about 225mOsm/kg to about 350 mOsm/kg, about 250 mOsm/kg to about 325 mOsm/kg,about 275 mOsm/kg to about 300 mOsm/kg, or about 285 mOsm/kg to about290 mOsm/kg.

A hydrogel material disclosed herein is typically resistant tobiodegradation upon administration to an individual. As used herein, theterm “resistant to biodegradation” is synonymous with “resistant tobioerosion”, “resistant to bioresorption”, “non-biodegradable”,“non-bioerodable” and “non-bioresorbable” and refers to a hydrogelmaterial disclosed herein that is not prone to degrading, eroding,resorbing, decomposing, or breaking down to any substantial orsignificant degree while implanted in an individual. This resistance tobiodegradation enables the hydrogel to retain and maintain the tissuespace in order to promote new tissue growth into the space. Non-limitingexamples of substantial non-degradation or resistance to biodegradationinclude less than 10% degradation of a hydrogel material over a timeperiod measured, less than 5% degradation of a hydrogel material over atime period measured, less than 3% degradation of a hydrogel materialover a time period measured, less than 1% degradation of a hydrogelmaterial over a time period measured. In an embodiment, a hydrogelmaterial disclosed herein is substantially non-biodegradable orresistant to biodegradation upon administration to an individual.

However, it is anticipated that as the space becomes filled withregenerated tissue which will replace the space being occupied by thehydrogel material. As such, the hydrogel material should be degradableor time. The rate of degradation imparted on the gel will be determinedby the speed of replacement with fat and maintenance of volume. In oneexample, hydrogel material will be resistant to degradation early onwith degradation occurring over several months.

In aspects of this embodiment, a hydrogel material is substantiallynon-biodegradable or resistance to biodegradation for, e.g., about 10days, about 20 days, about 30 days, about 40 days, about 50 days, about60 days, about 70 days, about 80 days, or about 90 days, beforebiodegradation occurs. In other aspects of this embodiment, a hydrogelmaterial is substantially non-biodegradable or resistant tobiodegradation for, e.g., at least 10 days, at least 20 days, at least30 days, at least 40 days, at least 50 days, at least 60 days, at least70 days, at least 80 days, or at least 90 days, before biodegradationoccurs. In yet other aspects of this embodiment, a hydrogel material issubstantially non-biodegradable or resistant to biodegradation for,e.g., at most 10 days, at most 20 days, at most 30 days, at most 40days, at most 50 days, at most 60 days, at most 70 days, at most 80days, or at most 90 days, before biodegradation occurs. In still otheraspects of this embodiment, a hydrogel material is substantiallynon-biodegradable or resistant to biodegradation for, e.g., about 10days to about 30 days, about 20 days to about 50 days, about 40 days toabout 60 days, about 50 days to about 80 days, or about 60 days to about90 days, before biodegradation occurs.

In aspects of this embodiment, biodegradation of a hydrogel materialdisclosed herein occurs with substantially first order release kineticsover a period of, e.g., about 7 days after, about 15 days, about 30days, about 45 days, about 60 days, about 75 days, or about 90 daysafter degradation begins. In other aspects of this embodiment,biodegradation of a hydrogel material disclosed herein occurs withsubstantially first order release kinetics over a period of e.g., atleast 7 days, at least 15 days, at least 30 days, at least 45 days, atleast 60 days, at least 75 days, or at least 90 days after degradationbegins. In yet other aspects of this embodiment, biodegradation of ahydrogel material disclosed herein occurs with substantially first orderrelease kinetics over a period of, e.g., about 10 days to about 30 days,about 20 days to about 50 days, about 40 days to about 60 days, about 50days to about 80 days, or about 60 days to about 90 days, afterdegradation begins.

The addition of a compound and hydrogel material to an adipose tissuemay be accomplished by any method that ensures sufficient distributionof the compound throughout the adipose tissue so as to promote formationof a blood supply sufficient to support the transplanted tissue. Forexample, mixing may occur through automated means, such as, e.g.,device-controlled agitation or centrifugation, or through manualmethods, such as, e.g., luer-locked syringes or vortexing.

The compositions disclosed herein may optionally comprise one or moreadditional materials useful as a filler for tissue space. Exemplaryfiller materials suitable with the compositions and methods disclosedherein include, without limitation, fillers comprising a polypeptidesuch as, e.g., a silk protein, (like silk fibroin), a resilin, aresilin-like polypeptide, an elastin, an elastin-like polypeptide, asilk protein-elastin-like polypeptide, an abductin, a byssus, a gliadin,a glutenin, abductin, keratin, gelatin, or collagen; fillers comprisinga polysaccharide such as, e.g., cellulose, agarose, chitosan, or chitin;and fillers comprising a polyester such as, e.g., D-lactic acid,L-lactic acid, racemic lactic acid, glycolic acid, caprolactone. Theseadditional tissue space fillers may be administered as a separatecomponent, or may be complexed with a hydrogel material disclosedherein.

The compositions disclosed herein may optionally comprise one or moreimmunosuppressive agents that reduce, and preferably prevent, rejectionof the transplanted tissue. As used herein, the term “immunosuppressiveagent” is synonymous with “immunosuppressive drug” and refers to acompound that inhibits or interferes with normal immune function.Exemplary immunosuppressive agents suitable with the compositions andmethods disclosed herein include, without limitation, agents thatinhibit T-cell/B-cell costimulation pathways like agents that interferewith the coupling of T-cells and B-cells via the CTLA4 and B7 pathways,including cyclosporine A, myophenylate mofetil, rapamicin, andanti-thymocyte globulin. An immunosuppressive drug is administered in aformulation that is compatible with the route of administration and isadministered to an individual at a dosage sufficient to achieve thedesired therapeutic effect.

The compositions disclosed herein may further and optionally compriseanother agent or combination of agents that provide a beneficial effectwhen the composition is administered to an individual. Such beneficialagents include, without limitation, an anti-itch agent, ananti-cellulite agent, an anti-scarring agent, an anti-inflammatoryagent, an anesthetic agent, an anti-irritant agent, a vasoconstrictor, avasodilator, an anti-hemorrhagic agent like a hemostatic agent oranti-fibrinolytic agent, a desquamating agent, a tensioning agent, ananti-acne agent, a pigmentation agent, an anti-pigmentation agent, or amoisturizing agent. Such agents are known in the art and described in,e.g., Gousse, et al., Heat Stable Hyaluronic Acid Compositions ForDermatological Use, U.S. patent application Ser. No. 12/714,337; Gousse,et al., Hydrogel Compositions Comprising Vasoconstricting andAnti-Hemorrhagic Agents for Dermatological Use, each of which is herebyincorporated by reference in its entirety.

In aspects of this embodiment, a composition disclosed herein comprisesan immunosuppressive agent, an anti-itch agent, an anti-cellulite agent,an anti-scarring agent, an anti-inflammatory agent, an anesthetic agent,an anti-irritant agent, a vasoconstrictor, a vasodilator, ananti-hemorrhagic agent like a hemostatic agent or anti-fibrinolyticagent, a desquamating agent, a tensioning agent, an anti-acne agent, apigmentation agent, an anti-pigmentation agent, and/or a moisturizingagent in an amount of, e.g., about 0.1%, about 0.2%, about 0.3%, about0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8% about 0.9%, about1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about7.0%, about 8.0%, about 9.0%, or about 10% by weight of the totalcomposition. In other aspects, a composition disclosed herein comprisesan immunosuppressive agent, an anti-itch agent, an anti-cellulite agent,an anti-scarring agent, an anti-inflammatory agent, an anesthetic agent,an anti-irritant agent, a vasoconstrictor, a vasodilator, ananti-hemorrhagic agent like a hemostatic agent or anti-fibrinolyticagent, a desquamating agent, a tensioning agent, an anti-acne agent, apigmentation agent, an anti-pigmentation agent, and/or a moisturizingagent in an amount of, e.g., at least 0.1%, at least 0.2%, at least0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, atleast 0.8% at least 0.9%, at least 1.0%, at least 2.0%, at least 3.0%,at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least8.0%, at least 9.0%, or at least 10% by weight of the total composition.In yet other aspects, a composition disclosed herein comprises animmunosuppressive agent, an anti-itch agent, an anti-cellulite agent, ananti-scarring agent, an anti-inflammatory agent, an anesthetic agent, ananti-irritant agent, a vasoconstrictor, a vasodilator, ananti-hemorrhagic agent like a hemostatic agent or anti-fibrinolyticagent, a desquamating agent, a tensioning agent, an anti-acne agent, apigmentation agent, an anti-pigmentation agent, and/or a moisturizingagent in an amount of, e.g., at most 0.1%, at most 0.2%, at most 0.3%,at most 0.4%, at most 0.5%, at most 0.6%, at most 0.7%, at most 0.8% atmost 0.9%, at most 1.0%, at most 2.0%, at most 3.0%, at most 4.0%, atmost 5.0%, at most 6.0%, at most 7.0%, at most 8.0%, at most 9.0%, or atmost 10% by weight of the total composition. In further aspects, acomposition disclosed herein comprises an anti-inflammatory agent in anamount of, e.g., about 0.1% to about 0.5%, about 0.1% to about 1.0%,about 0.1% to about 2.0%, about 0.1% to about 3.0%, about 0.1% to about4.0%, about 0.1% to about 5.0%, about 0.2% to about 0.9%, about 0.2% toabout 1.0%, about 0.2% to about 2.0%, about 0.5% to about 1.0%, or about0.5% to about 2.0% by weight of the total composition.

In other aspects of this embodiment, a composition disclosed hereincomprises an immunosuppressive agent, an anti-itch agent, ananti-cellulite agent, an anti-scarring agent, an anti-inflammatoryagent, an anesthetic agent, an anti-irritant agent, a vasoconstrictor, avasodilator, an anti-hemorrhagic agent like a hemostatic agent oranti-fibrinolytic agent, a desquamating agent, a tensioning agent, ananti-acne agent, a pigmentation agent, an anti-pigmentation agent,and/or a moisturizing agent at a concentration of, e.g., about 0.01mg/mL, about 0.02 mg/mL, about 0.03 mg/mL, about 0.04 mg/mL, about 0.05mg/mL, about 0.06 mg/mL, about 0.07 mg/mL, about 0.08 mg/mL, about 0.09mg/mL, about 0.1 mg/mL, about 0.2 mg/mL, about 0.3 mg/mL, about 0.4mg/mL, about 0.5 mg/mL, about 0.6 mg/mL, about 0.7 mg/mL, about 0.8mg/mL, about 0.9 mg/mL, about 1.0 mg/mL, about 2.0 mg/mL, about 3.0mg/mL, about 4.0 mg/mL, about 5.0 mg/mL, about 6.0 mg/mL, about 7.0mg/mL, about 8.0 mg/mL, about 9.0 mg/mL, or about 10 mg/mL. In yet otheraspects of this embodiment, a composition disclosed herein comprises animmunosuppressive agent, an anti-itch agent, an anti-cellulite agent, ananti-scarring agent, an anti-inflammatory agent, an anesthetic agent, ananti-irritant agent, a vasoconstrictor, a vasodilator, ananti-hemorrhagic agent like a hemostatic agent or anti-fibrinolyticagent, a desquamating agent, a tensioning agent, an anti-acne agent, apigmentation agent, an anti-pigmentation agent, and/or a moisturizingagent at a concentration of, e.g., at least 0.01 mg/mL, at least 0.02mg/mL, at least 0.03 mg/mL, at least 0.04 mg/mL, at least 0.05 mg/mL, atleast 0.06 mg/mL, at least 0.07 mg/mL, at least 0.08 mg/mL, at least0.09 mg/mL, at least 0.1 mg/mL, at least 0.2 mg/mL, at least 0.3 mg/mL,at least 0.4 mg/mL, at least 0.5 mg/mL, at least 0.6 mg/mL, at least 0.7mg/mL, at least 0.8 mg/mL, at least 0.9 mg/mL, at least 1.0 mg/mL, atleast 2.0 mg/mL, at least 3.0 mg/mL, at least 4.0 mg/mL, at least 5.0mg/mL, at least 6.0 mg/mL, at least 7.0 mg/mL, at least 8.0 mg/mL, atleast 9.0 mg/mL, or at least 10 mg/mL. In still other aspects of thisembodiment, a composition disclosed herein comprises animmunosuppressive agent, an anti-itch agent, an anti-cellulite agent, ananti-scarring agent, an anti-inflammatory agent, an anesthetic agent, ananti-irritant agent, a vasoconstrictor, a vasodilator, ananti-hemorrhagic agent like a hemostatic agent or anti-fibrinolyticagent, a desquamating agent, a tensioning agent, an anti-acne agent, apigmentation agent, an anti-pigmentation agent, and/or a moisturizingagent at a concentration of, e.g., at most 0.01 mg/mL, at most 0.02mg/mL, at most 0.03 mg/mL, at most 0.04 mg/mL, at most 0.05 mg/mL, atmost 0.06 mg/mL, at most 0.07 mg/mL, at most 0.08 mg/mL, at most 0.09mg/mL, at most 0.1 mg/mL, at most 0.2 mg/mL, at most 0.3 mg/mL, at most0.4 mg/mL, at most 0.5 mg/mL, at most 0.6 mg/mL, at most 0.7 mg/mL, atmost 0.8 mg/mL, at most 0.9 mg/mL, at most 1.0 mg/mL, at most 2.0 mg/mL,at most 3.0 mg/mL, at most 4.0 mg/mL, at most 5.0 mg/mL, at most 6.0mg/mL, at most 7.0 mg/mL, at most 8.0 mg/mL, at most 9.0 mg/mL, or atmost 10 mg/mL. In further aspects, a composition disclosed hereincomprises an anti-inflammatory agent at a concentration of, e.g., about0.01 mg/mL to about 0.7 mg/mL, about 0.06 mg/mL to about 0.7 mg/mL,about 0.01 mg/mL to about 1.0 mg/mL, about 0.05 mg/mL to about 1.0mg/mL, about 0.06 mg/mL to about 1.0 mg/mL, about 0.1 mg/mL to about 1.0mg/mL, about 0.1 mg/mL to about 2.0 mg/mL, about 0.1 mg/mL to about 3.0mg/mL, about 0.1 mg/mL to about 4.0 mg/mL, about 0.1 mg/mL to about 5.0mg/mL, about 0.2 mg/mL to about 0.9 mg/mL, about 0.2 mg/mL to about 1.0mg/mL, about 0.2 mg/mL to about 2.0 mg/mL, about 0.5 mg/mL to about 1.0mg/mL, or about 0.5 mg/mL to about 2.0 mg/mL.

In another embodiment, a composition disclosed herein does not comprisean immunosuppressive agent, an anti-itch agent, an anti-cellulite agent,an anti-scarring agent, an anti-inflammatory agent, an anesthetic agent,an anti-irritant agent, a vasoconstrictor, a vasodilator, ananti-hemorrhagic agent like a hemostatic agent or anti-fibrinolyticagent, a desquamating agent, a tensioning agent, an anti-acne agent, apigmentation agent, an anti-pigmentation agent, and/or a moisturizingagent.

A compound disclosed herein, or a composition comprising such a compoundis generally administered to an individual as a pharmaceuticalcomposition. Pharmaceutical compositions may be prepared by combining atherapeutically effective amount of at least one compound according tothe present specification, or a pharmaceutically acceptable acidaddition salt thereof, as an active ingredient, with conventionalacceptable pharmaceutical excipients, and by preparation of unit dosageforms suitable for topical ocular use. The therapeutically efficientamount typically is between about 0.0001% (w/v) and about 5% (w/v),preferably about 0.001% (w/v) to about 1.0% (w/v) in liquidformulations. As used herein, the term “pharmaceutical composition”refers to a therapeutically effective concentration of an activecompound, such as, e.g., any of the compounds disclosed herein.Preferably, the pharmaceutical composition does not produce an adverse,allergic, or other untoward or unwanted reaction when administered to anindividual. A pharmaceutical composition disclosed herein is useful formedical and veterinary applications. A pharmaceutical composition may beadministered to an individual alone, or in combination with othersupplementary active compounds, agents, drugs or hormones. Thepharmaceutical compositions may be manufactured using any of a varietyof processes, including, without limitation, conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilizate,tablet, pill, pellet, capsule, powder, syrup, elixir, or any otherdosage form suitable for administration.

The compounds disclosed herein may also be incorporated into a drugdelivery platform in order to achieve a controlled compound releaseprofile over time. Such a drug delivery platform comprises a compounddisclosed herein dispersed within a polymer matrix, typically abiodegradable, bioerodible, and/or bioresorbable polymer matrix. As usedherein, the term “polymer” refers to synthetic homo- or copolymers,naturally occurring homo- or copolymers, as well as syntheticmodifications or derivatives thereof having a linear, branched or starstructure. Copolymers can be arranged in any form, such as, e.g.,random, block, segmented, tapered blocks, graft, or triblock. Polymersare generally condensation polymers. Polymers can be further modified toenhance their mechanical or degradation properties by introducingcross-linking agents or changing the hydrophobicity of the sideresidues. If crosslinked, polymers are usually less than 5% crosslinked,usually less than 1% crosslinked.

Suitable polymers for use in a drug delivery platform include, withoutlimitation, alginates, aliphatic polyesters, polyalkylene oxalates,polyamides, polyamidoesters, polyanhydrides, polycarbonates, polyesters,polyethylene glycol, polyhydroxyaliphatic carboxylic acids,polyorthoesters, polyoxaesters, polypeptides, polyphosphazenes,polysaccharides, and polyurethanes. The polymer usually comprises atleast about 10% (w/w), at least about 20% (w/w), at least about 30%(w/w), at least about 40% (w/w), at least about 50% (w/w), at leastabout 60% (w/w), at least about 70% (w/w), at least about 80% (w/w), orat least about 90% (w/w) of the drug delivery platform. Examples ofbiodegradable, bioerodible, and/or bioresorbable polymers and methodsuseful to make a drug delivery platform are described in, e.g., Drost,et. al., Controlled Release Formulation, U.S. Pat. No. 4,756,911; Smith,et. al., Sustained Release Drug Delivery Devices, U.S. Pat. No.5,378,475; Wong and Kochinke, Formulation for Controlled Release ofDrugs by Combining Hyrophilic and Hydrophobic Agents, U.S. Pat. No.7,048,946; Hughes, et. Al., Compositions and Methods for LocalizedTherapy of the Eye, U.S. Patent Publication 2005/0181017; Hughes,Hypotensive Lipid-Containing Biodegradable Intraocular Implants andRelated Methods, U.S. Patent Publication 2005/0244464; Altman, et al.,Silk Fibroin Hydrogels and Uses Thereof, U.S. patent application Ser.No. 12/764,039, filed on Apr. 20, 2010; each of which is incorporated byreference in its entirety.

In aspects of this embodiment, a polymer composing the matrix is apolypeptide such as, e.g., silk fibroin, keratin, or collagen. In otheraspects of this embodiment, a polymer composing the matrix is apolysaccharide such as, e.g., cellulose, agarose, elastin, chitosan,chitin, or a glycosaminoglycan like chondroitin sulfate, dermatansulfate, keratan sulfate, or hyaluronic acid (as discussed supra). Inyet other aspects of this embodiment, a polymer composing the matrix isa polyester such as, e.g., D-lactic acid, L-lactic acid, racemic lacticacid, glycolic acid, caprolactone, and combinations thereof.

One of ordinary skill in the art appreciates that the selection of asuitable polymer for forming a suitable disclosed drug delivery platformdepends on several factors. The more relevant factors in the selectionof the appropriate polymer(s), include, without limitation,compatibility of polymer with a drug, desired release kinetics of adrug, desired biodegradation kinetics of platform at implantation site,desired bioerodible kinetics of platform at implantation site, desiredbioresorbable kinetics of platform at implantation site, in vivomechanical performance of platform, processing temperatures,biocompatibility of platform, and patient tolerance. Other relevantfactors that, to some extent, dictate the in vitro and in vivo behaviorof the polymer include the chemical composition, spatial distribution ofthe constituents, the molecular weight of the polymer and the degree ofcrystallinity.

A drug delivery platform includes both a sustained release drug deliveryplatform and an extended release drug delivery platform. As used herein,the term “sustained release” refers to the release of a compounddisclosed herein over a period of about seven days or more. As usedherein, the term “extended release” refers to the release of a compounddisclosed herein over a period of time of less than about seven days.

In aspects of this embodiment, a sustained release drug deliveryplatform releases a compound disclosed herein with substantially firstorder release kinetics over a period of, e.g., about 7 days afteradministration, about 15 days after administration, about 30 days afteradministration, about 45 days after administration, about 60 days afteradministration, about 75 days after administration, or about 90 daysafter administration. In other aspects of this embodiment, a sustainedrelease drug delivery platform releases a compound disclosed herein withsubstantially first order release kinetics over a period of, e.g., atleast 7 days after administration, at least 15 days afteradministration, at least 30 days after administration, at least 45 daysafter administration, at least 60 days after administration, at least 75days after administration, or at least 90 days after administration.

In aspects of this embodiment, a drug delivery platform releases acompound disclosed herein with substantially first order releasekinetics over a period of, e.g., about 1 day after administration, about2 days after administration, about 3 days after administration, about 4days after administration, about 5 days after administration, or about 6days after administration. In other aspects of this embodiment, a drugdelivery platform releases a compound disclosed herein withsubstantially first order release kinetics over a period of, e.g., atmost 1 day after administration, at most 2 days after administration, atmost 3 days after administration, at most 4 days after administration,at most 5 days after administration, or at most 6 days afteradministration.

A pharmaceutical composition disclosed herein can optionally include apharmaceutically acceptable carrier that facilitates processing of anactive compound into pharmaceutically acceptable compositions. As usedherein, the term “pharmacologically acceptable carrier” is synonymouswith “pharmacological carrier” and refers to any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, additive, auxiliary, or excipient.” Such acarrier generally is mixed with an active compound or permitted todilute or enclose the active compound and can be a solid, semi-solid, orliquid agent. It is understood that the active compounds can be solubleor can be delivered as a suspension in the desired carrier or diluent.Any of a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., starch, magnesium stearate, mannitol, sodium saccharin, talcum,cellulose, glucose, sucrose, lactose, trehalose, magnesium carbonate,and the like; solvents; dispersion media; coatings; antibacterial andantifungal agents; isotonic and absorption delaying agents; or any otherinactive ingredient. Selection of a pharmacologically acceptable carriercan depend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activecompound, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in Pharmaceutical Dosage Forms andDrug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams& Wilkins Publishers, 7^(th) ed. 1999); Remington: The Science andPractice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams &Wilkins, 20^(th) ed. 2000); Goodman & Gilman's The Pharmacological Basisof Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional,10^(th) ed. 2001); and Handbook of Pharmaceutical Excipients (Raymond C.Rowe et al., APhA Publications, 4^(th) edition 2003). These protocolsare routine and any modifications are well within the scope of oneskilled in the art and from the teaching herein.

A pharmaceutical composition disclosed herein may optionally include,without limitation, other pharmaceutically acceptable components (orpharmaceutical components), including, without limitation, buffers,preservatives, tonicity adjusters, salts, antioxidants, osmolalityadjusting agents, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed herein,provided that the resulting preparation is pharmaceutically acceptable.Such buffers include, without limitation, acetate buffers, boratebuffers, citrate buffers, phosphate buffers, neutral buffered saline,and phosphate buffered saline. It is understood that acids or bases canbe used to adjust the pH of a composition as needed. Pharmaceuticallyacceptable antioxidants include, without limitation, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole, and butylated hydroxytoluene. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilizedoxy chloro composition, such as, e.g., sodium chlorite and chelants,such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, andCaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. The pharmaceuticalcomposition may be provided as a salt and can be formed with many acids,including but not limited to, hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents than are the corresponding free base forms.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition useful inthe invention.

Aspects of the present specification provide, in part, a method oftreating a soft tissue condition of an individual by administering acomposition disclosed herein. As used herein, the term “treating,”refers to reducing or eliminating in an individual a cosmetic orclinical symptom of a soft tissue condition characterized by a softtissue imperfection, defect, disease, and/or disorder; or delaying orpreventing in an individual the onset of a cosmetic or clinical symptomof a condition characterized by a soft tissue imperfection, defect,disease, and/or disorder. For example, the term “treating” can meanreducing a symptom of a condition characterized by a soft tissue defect,disease, and/or disorder by, e.g., at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90% or at least 100%. The effectiveness of a compound disclosed hereinin treating a condition characterized by a soft tissue defect, disease,and/or disorder can be determined by observing one or more cosmetic,clinical symptoms, and/or physiological indicators associated with thecondition. An improvement in a soft tissue defect, disease, and/ordisorder also can be indicated by a reduced need for a concurrenttherapy. Those of skill in the art will know the appropriate symptoms orindicators associated with specific soft tissue defect, disease, and/ordisorder and will know how to determine if an individual is a candidatefor treatment with a compound or composition disclosed herein.

A composition or compound is administered to an individual. Anindividual is typically a human being. Typically, any individual who isa candidate for a conventional soft tissue replacement procedure is acandidate for a soft tissue replacement procedure disclosed herein. Inaddition, the presently disclosed compositions and methods may apply toindividuals seeking a small/moderate enlargement, shape change orcontour alteration of a body part or region, which may not betechnically possible or aesthetically acceptable with existing softtissue implant technology. Pre-operative evaluation typically includesroutine history and physical examination in addition to thoroughinformed consent disclosing all relevant risks and benefits of theprocedure.

The composition and methods disclosed herein are useful in treating asoft tissue condition. A soft tissue condition includes, withoutlimitation, a soft tissue imperfection, defect, disease, and/ordisorder. Non-limiting examples of a soft tissue condition includebreast imperfection, defect, disease and/or disorder, such as, e.g., abreast augmentation, a breast reconstruction mastopexy, micromastia,thoracic hypoplasia, Poland's syndrome, defects due to implantcomplications like capsular contraction and/or rupture; a facialimperfection, defect, disease or disorder, such as, e.g., a facialaugmentation, a facial reconstruction, Parry-Romberg syndrome, lupuserythematosus profundus, dermal divots, sunken cheeks, thin lips, nasalimperfections or defects, retro-orbital imperfections or defects, afacial fold, line and/or wrinkle like a glabellar line, a nasolabialline, a perioral line, and/or a marionette line, and/or other contourdeformities or imperfections of the face; a neck imperfection, defect,disease or disorder; a skin imperfection, defect, disease and/ordisorder; other soft tissue imperfections, defects, diseases and/ordisorders, such as, e.g., an augmentation or a reconstruction of theupper arm, lower arm, hand, shoulder, back, torso including abdomen,buttocks, upper leg, lower leg including calves, foot including plantarfat pad, eye, genitals, or other body part, region or area, or a diseaseor disorder affecting these body parts, regions or areas; urinaryincontinence, fecal incontinence, other forms of incontinence; andgastroesophageal reflux disease (GERD).

The amount of adipose tissue and/or hydrogel material used with any ofthe methods as disclosed herein will typically be determined based onthe alteration and/or improvement desired, the reduction and/orelimination of a soft tissue condition symptom desired, the clinicaland/or cosmetic effect desired by the individual and/or physician, andthe body part or region being treated. The effectiveness of adiposetissue and/or hydrogel material administration may be manifested by oneor more of the following clinical and/or cosmetic measures: alteredand/or improved soft tissue shape, altered and/or improved soft tissuesize, altered and/or improved soft tissue contour, altered and/orimproved tissue function, improved transplant tissue survival, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign material.

For example, for breast augmentation procedures, effectiveness ofadipose tissue and/or hydrogel material administration may be manifestedby one or more of the following clinical and/or cosmetic measures:increased breast size, altered breast shape, altered breast contour,sustained engraftment, decreased rate of liponecrotic cyst formation,improved patient satisfaction and/or quality of life, and decreased useof breast implant.

As another example, effectiveness of adipose tissue and/or hydrogelmaterial administration in treating a facial soft tissue may bemanifested by one or more of the following clinical and/or cosmeticmeasures: increased size, shape, and/or contour of facial feature likeincreased size, shape, and/or contour of lip, cheek or eye region;altered size, shape, and/or contour of facial feature like altered size,shape, and/or contour of lip, cheek or eye region shape; reduction orelimination of a wrinkle, fold or line in the skin; resistance to awrinkle, fold or line in the skin; rehydration of the skin; increasedelasticity to the skin; reduction or elimination of skin roughness;increased and/or improved skin tautness; reduction or elimination ofstretch lines or marks; increased and/or improved skin tone, shine,brightness and/or radiance; increased and/or improved skin color,reduction or elimination of skin paleness; sustained engraftment ofcomposition; decreased side effects; improved patient satisfactionand/or quality of life.

As yet another example, for urinary incontinence procedures,effectiveness of adipose tissue and/or hydrogel material administrationfor sphincter support may be manifested by one or more of the followingclinical measures: decreased frequency of incontinence, sustainedengraftment, improved patient satisfaction and/or quality of life, anddecreased use of implantable foreign filler.

The amount of a compound used with any of the methods disclosed hereinwill typically be a therapeutically effective amount. As used herein,the term “therapeutically effective amount” is synonymous with“effective amount”, “therapeutically effective dose”, and/or “effectivedose” and refers to the amount of compound that will elicit thebiological, cosmetic or clinical response being sought by thepractitioner in an individual in need thereof. As a non-limitingexample, an effective amount is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. As another non-limiting example, an effective amount is anamount sufficient to promote formation of new blood vessels andassociated vasculature (angiogenesis) and/or an amount sufficient topromote repair or remodeling of existing blood vessels and associatedvasculature. The appropriate effective amount to be administered for aparticular application of the disclosed methods can be determined bythose skilled in the art, using the guidance provided herein. Forexample, an effective amount can be extrapolated from in vitro and invivo assays as described in the present specification. One skilled inthe art will recognize that the condition of the individual can bemonitored throughout the course of therapy and that the effective amountof a compound or composition disclosed herein that is administered canbe adjusted accordingly.

In aspects of this embodiment, the amount of a compound added is, e.g.,0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg,500 mg, 750 mg, or 1000 mg. In other aspects of this embodiment, theamount of a compound added is, e.g., 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL,0.5 mg/mL, 1 mg/mL, 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 150 mg/mL, 200mg/mL, 250 mg/mL, 500 mg/mL, 750 mg/mL, or 1000 mg/mL. In yet otheraspects of this embodiment, the amount of a compound added is, e.g.,0.01 mg/10 mL of tissue, 0.05 mg/10 mL of tissue, 0.1 mg/10 mL oftissue, 0.5 mg/10 mL of tissue, 1 mg/10 mL of tissue, 5 mg/10 mL oftissue, 10 mg/10 mL of tissue, 20 mg/10 mL of tissue, 30 mg/10 mL oftissue, 40 mg/10 mL of tissue, 50 mg/10 mL of tissue, 60 mg/10 mL oftissue, 70 mg/10 mL of tissue, 80 mg/10 mL of tissue, 90 mg/10 mL oftissue, 100 mg/10 mL of tissue, 150 mg/10 mL of tissue, 200 mg/10 mL oftissue, 250 mg/10 mL of tissue, 500 mg/10 mL of tissue, 750 mg/10 mL oftissue, or 1000 mg/10 mL of tissue.

In aspects of this embodiment, the amount of a compound added is, e.g.,about 0.01 mg to about 0.1 mg, about 0.1 mg to about 1 mg, about 1 mg toabout 10 mg, about 10 mg to about 100 mg, or about 100 mg to about 1000mg. In other aspects of this embodiment, the amount of a compound addedis, e.g., about 0.01 mg/mL to about 0.1 mg/mL, about 0.1 mg/mL to about1 mg/mL, about 1 mg/mL to about 10 mg/mL, about 10 mg/mL to about 100mg/mL, or about 100 mg/mL to about 1000 mg/mL. In yet other aspects ofthis embodiment, the amount of a compound added is, e.g., about 0.01mg/10 mL of tissue to about 0.1 mg/10 mL of tissue, about 0.1 mg/10 mLof tissue to about 1 mg/10 mL of tissue, about 1 mg/10 mL of tissue toabout 10 mg/10 mL of tissue, about 10 mg/10 mL of tissue to about 100mg/10 mL of tissue, or about 100 mg/10 mL of tissue to about 1000 mg/10mL of tissue.

In aspects of this embodiment, the amount of a compound added is, e.g.,about 0.0001% (w/v) to about 5% (w/v), about 0.001% (w/v) to about 5%(w/v), about 0.01% (w/v) to about 5% (w/v), about 0.1% (w/v) to about 5%(w/v), or about 1% (w/v) to about 5% (w/v). In other aspects of thisembodiment, the amount of a compound added is, e.g., about 0.001% (w/v)to about 1.0% (w/v), about 0.01% (w/v) to about 1.0% (w/v), or about0.1% (w/v) to about 1.0% (w/v).

A composition disclosed herein can be mixed with the harvested adiposetissue and, as such, be administered simultaneously with the adiposetissue. Additionally, or alternatively, a composition disclosed hereincan be administered after administration of the adipose tissue. Inaspects of this embodiment, a composition disclosed herein isadministered to the individual additionally, or alternatively, e.g.,about 1 day after adipose tissue implantation, about 2 days afteradipose tissue implantation, about 3 days after adipose tissueimplantation, or about 4 days after adipose tissue implantation. Inother aspects, a composition disclosed herein is administered to theindividual additionally, or alternatively, e.g., about once a day forabout 3 to about 10 consecutive days, about once a day for about 7 toabout 14 consecutive days, about once a day for about 10 to about 21consecutive days, or about once a day for about 14 to about 28consecutive days. In other aspects, the daily dose can be, e.g., twice aday, three times a day, or four times a day.

The route of administration of composition administered to an individualpatient will typically be determined based on the cosmetic and/orclinical effect desired by the individual and/or physician and the bodypart or region being treated. Compositions or adipose tissue may beadministered by any means known to persons of ordinary skill in the artincluding, without limitation, syringe with needle, catheter, or bydirect surgical implantation. In addition, the disclosed composition oradipose tissue can be administered once, or over a plurality of times.Ultimately, the timing used will follow quality care standards.

For a breast soft tissue replacement procedure, the route ofadministration may include axillary, periareolar, and/or inframammaryroutes. For a facial soft tissue replacement procedure, the route ofadministration can be frontal, temporal, zygomatic, periocular,amdibula, perioral or chin routes. In urinary incontinence procedures,the route of administration may include transurethral or periurethralroutes. Alternatively or in addition, the transplant may be deliveredvia an antegrade route. The routes discussed herein do not exclude theuse of multiple routes to achieve the desired clinical effect, orumbilical incision. Alternatively or in addition, cell-enhanced tissuemay be delivered through a transaxillary endoscopic subpectoralapproach.

A composition or compound administered to an individual to treat a softtissue condition promotes formation of a blood supply sufficient tosupport the transplanted tissue. Blood supply formation includes,without limitation, formation of new blood vessels and associatedvasculature (angiogenesis) and repair or remodel of existing bloodvessels and associated vasculature.

Aspects of the present specification disclose, in part, a method oftreating a soft tissue condition of an individual, the method comprisingthe steps of a) administering the adipose tissue to a site of the softtissue condition; and b) administering a composition comprising acompound as disclosed herein to the site of the soft tissue condition.The adipose tissue and compound(s) administered include the adiposetissue disclosed herein. In these methods, the adipose tissue isadministered separately from a composition comprising the compound.Thus, in one aspect of this embodiment, adipose tissue is administeredfirst, followed by administration of a composition comprising thecompound. In another aspect of this embodiment, a composition comprisinga compound is administered first, followed by administration of adiposetissue. In certain embodiments, the composition comprising a compoundfurther comprises an HA. In other embodiments, a second compositioncomprising HA is administered at the same time, before, or after theadministration of the first composition that comprises one or more ofthe compounds disclosed herein.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification, including those pertaining to the compositions andmethods of soft tissue replacement disclosed herein.

Example 1 Synthesis of Compounds of Formula I

This example illustrates the synthesis scheme for the compounds offormula I.

The methods of preparing the compounds disclosed herein are furtherillustrated by the following non-limiting examples, which are summarizedin the reaction schemes of FIGS. 1-9 wherein the compounds areidentified by the same designator in both the Examples and the Figures.

2-Alkyl-cyclopentane-1,3-dione (1a, FIG. 1). A mixture of1,3-cyclopentanedione (89.4 mmol, Aldrich), I-R² (96.4 mmol, Aldrich),and KOH (5.097 g, 90.8 mmol) in water (25 mL)/dioxane (75 mL) was heatedat reflux. After 5 hours, a solution of KOH (2 g) and I-R² (2 mmol) inwater (5 mL)/dioxane (15 mL) was added and after another 3 hours atreflux, the solution was stirred at room temperature overnight. Asolution of KOH (2 g) and I-R² (2.4 mmol) in water (5 mL)/dioxane (15mL) was added to the overnight reaction and heating at reflux. After 4hours, the mixture was cooled to room temperature and extracted withether (1×100 mL, 3×75 mL). The combined ether extracts were evaporated,the residue combined with 10% HCl (50 mL), and the resulting mixtureplaced in a 120° C. oil bath until boiling was observed (ca. 15minutes). The mixture was cooled to room temperature, neutralized byaddition of NaHCO₃ solution (150 mL, saturated) and the resultingmixture extracted with CH₂Cl₂ (4×75 mL). The combined CH₂Cl₂ solutionwas dried (MgSO₄), filtered and evaporated to leave a brown oil whichwas used directly in the next step.

2-Alkyl-2-methyl-cyclopentane-1,3-dione (2a, FIG. 1). A mixture of2-methyl-1,3-cyclopentanedione (10.025 g, 89.4 mmol, Aldrich), I-R²(96.4 mmol, Aldrich), and KOH (5.097 g, 90.8 mmol) in water (25mL)/dioxane (75 mL) was heated at reflux. After 5 hours, a solution ofKOH (2 g) and I-R² (2 mmol) in water (5 mL)/dioxane (15 mL) was addedand after another 3 hours at reflux, the solution was stirred overnightat room temperature. A solution of KOH (2 g) and I-R² (2.4 mmol) inwater (5 mL)/dioxane (15 mL) was added to the overnight reaction andheating at reflux. After 4 hours, the mixture was cooled to roomtemperature and extracted with ether (1×100 mL, 3×75 mL). The combinedether extracts were evaporated, the residue combined with 10% HCl (50mL), and the resulting mixture placed in a 120° C. oil bath until itbegan boiling (ca. 15 minutes). The mixture was cooled to roomtemperature, neutralized by addition of NaHCO₃ solution (150 mL,saturated) and the resulting mixture extracted with CH₂Cl₂ (4×75 mL).The combined CH₂Cl₂ solution was dried (MgSO₄), filtered and evaporatedto leave a brown oil which was used directly in the next step.

2,2-Dialkyl-methyl-cyclopentane-1,3-dione (2b, FIG. 1). A mixture of2-alkyl-1,3-cyclopentanedione 1a (89.4 mmol, Aldrich), I-R³ (96.4 mmol,Aldrich), and KOH (5.097 g, 90.8 mmol) in water (25 mL)/dioxane (75 mL)was heated at reflux. After 5 hours, a solution of KOH (2 g) and I-R³ (2mmol) in water (5 mL)/dioxane (15 mL) was added and after another 3hours at reflux, the solution was stirred at room temperature overnight.A solution of KOH (2 g) and I-R³ (2.4 mmol) in water (5 mL)/dioxane (15mL) was added to the overnight reaction and heating at reflux. After 4hours, the mixture was cooled to room temperature and extracted withether (1×100 mL, 3×75 mL). The combined ether extracts were evaporated,the residue combined with 10% HCl (50 mL), and the resulting mixtureplaced in a 120° C. oil bath until it began boiling (ca. 15 minutes).The mixture was then cooled to room temperature, neutralized by additionof NaHCO₃ solution (150 mL, saturated) and the resulting mixtureextracted with CH₂Cl₂ (4×75 mL). The combined CH₂Cl₂ solution was dried(MgSO₄), filtered and evaporated to leave a brown oil which was useddirectly in the next step.

Spiro[2.4]heptane-4,7-dione (2c, FIG. 1). A mixture of2-alkyl-1,3-cyclopentanedione 1a (89.4 mmol, Aldrich), 1,2-dibromoethane(120 mmol, Aldrich), and KOH (5.097 g, 90.8 mmol) in water (25mL)/dioxane (75 mL) was heated at reflux for 24 hours. The mixture wascooled, and the product extracted with ether (1×100 mL, 3×75 mL). Thecombined ether extracts were evaporated, the residue combined with 10%HCl (50 mL), and the resulting mixture placed in a 120° C. oil bathuntil boiling was observed (ca. 15 minutes). The mixture was then cooledto room temperature, neutralized by addition of NaHCO₃ solution (150 mL,saturated) and the resulting mixture extracted with CH₂Cl₂ (4×75 mL).The combined CH₂Cl₂ solution was dried (MgSO₄), filtered and evaporatedto leave a brown oil which was used directly in the next step.

2,2-Dimethyl-cyclopentane-1,3-dione (2, FIG. 1). Synthesized accordingto Agosta and Smith, J. Org. Chem., 35: 3856 (1970). A mixture of2-methyl-1,3-cyclopentanedione (10.025 g, 89.4 mmol, Aldrich), methyliodide (6.0 mL, 96.4 mmol, Aldrich), and KOH (5.097 g, 90.8 mmol) inwater (25 mL)/dioxane (75 mL) was heated at reflux. After 5 hours, asolution of KOH (2 g) and MeI (2.4 mL) in water (5 mL)/dioxane (15 mL)was added and after another 3 hours at reflux, the solution was stirredat room temperature overnight. A solution of KOH (2 g) and MeI (2.4 mL)in water (5 mL)/dioxane (15 mL) was added to the overnight reaction andheating at reflux. After 4 hours, the mixture was cooled to roomtemperature and extracted with ether (1×100 mL, 3×75 mL). The combinedether extracts were evaporated, the residue combined with 10% HCl (50mL), and the resulting mixture placed in a 120° C. oil bath until itbegan boiling (ca. 15 minutes). The mixture was cooled to roomtemperature, neutralized by addition of saturated NaHCO₃ solution (150mL) and the resulting mixture extracted with CH₂Cl₂ (4×75 mL). Thecombined CH₂Cl₂ solution was dried (MgSO₄), filtered and evaporated toleave a brown oil (10.474 g, 83 mmol, 93%) which was used directly inthe next step.

(S)-3-Hydroxy-2,2-dimethyl-cyclopentanone (3, FIG. 2). Synthesizedaccording to Brooks, et al., J. Org. Chem., 52: 3223 (1987). A 35° C.(internal temperature) solution of D-glucose (106.73 g, 592 mmol,Aldrich) in water (690 mL) in a 4 L Erlenmeyer was treated with baker'syeast (71.065 g, Fleischmann's). The mixture was fermented for 2 hours,and 2,2-dimethyl-cyclopentane-1,3-dione (2) (7.316 g, 58 mmol) wasadded. The mixture was stirred for 48 hours and filtered through celite,washing with about 1 L CH₂Cl₂. About 100 mL of brine was added to thefiltrate and the layers separated using a separatory funnel. Brine (400mL) was added to the aqueous layer and the resulting solution extractedfurther with CH₂Cl₂ (3×500 mL). The combined CH₂Cl₂ solution was dried(MgSO₄), filtered and evaporated to leave a yellow oil. Flashchromatography (11×5 cm, 20% EtOAc/hexs→25%→30%→40%→50%) gave alcohol 3(2.435 g, 19 mmol, 33%).

The enantiomeric excess of 3 was assayed by ¹H NMR of the correspondingMosher's ester which was prepared by treatment of alcohol 3 (11 mg, 0.09mmol) in dichloroethane (0.3 mL, Aldrich) with pyridine (27 μL, 0.33mmol, Aldrich) and (R)-α-methoxy-α-trifluoromethyphenylacetic acidchloride (58 μL, 0.31 mmol, Fluka). The mixture was stirred overnightand then partitioned between water (10 mL) and ether (10 mL). The etherlayer was washed with 1 M HCl (10 mL) and saturated NaHCO₃ solution andthen dried (MgSO₄), filtered and evaporated. ¹H NMR analysis was done onthe crude ester.

(S)-3-(tert)-Butyl-dimethyl-silanyloxy-2,2-dimethyl-cyclopentanone (4,FIG. 2). A solution of alcohol (3) (520 mg, 4.1 mmol) and 2,6-lutidine(0.56 mL, 4.8 mmol, Aldrich) in CH₂Cl₂ (8.0 mL, Aldrich) was treatedwith TBSOTf (1.0 mL, 4.3 mmol, Aldrich). After 5.5 hours, saturatedNaHCO₃ solution (20 mL) was added and the mixture extracted with CH₂Cl₂(20 mL). The CH₂Cl₂ solution was washed with 20 mL each of 1 M HCl,saturated NaHCO₃ solution, and brine and then dried (MgSO₄), filteredand evaporated. Flash chromatography (5×5 cm, 10% Et₂O/pentane) gave TBSether (4) (698 mg, 2.9 mmol, 70%).

(S)-3-(tert)-Butyl-dimethyl-silanyloxy-2,2-dimethyl-5-phenylselanyl-cyclopentanone(5, FIG. 2). A solution of TBS ether (4) (1.496 g, 6.2 mmol) in THF (2mL, Aldrich) was added dropwise to a −78° C. solution of LDA (4.9 mL,7.3 mmol, 1.5 M/cyclohexane, Aldrich) in THF (22 mL, Aldrich), rinsingwith 2 mL THF. After 15 minutes, a solution of PhSeCl (1.424 g, 7.4mmol, Aldrich) in THF (2 mL) was quickly added by cannula, rinsing with2 mL THF. The solution was stirred for 10 minutes and then partitionedbetween 50 mL 0.5 M HCl and 75 mL ether. The ether layer was washed with30 mL each of water, saturated NaHCO₃ solution, and brine and then dried(MgSO₄), filtered and evaporated. Flash chromatography (2%EtOAc/hexs→4%) gave phenylselenide (5) (1.641 g, 4.1 mmol, 67%) alongwith 476 mg of mixed fractions containing a lower R_(f) impurity.

(S)-4-(tert)-Butyl-dimethyl-silanyloxy-5,5-dimethyl-cyclopent-2-enone(6, FIG. 2). A solution of selenide (5) (1.641 g, 4.1 mmol) and pyridine(0.62 mL, 7.7 mmol, Aldrich) in CH₂Cl₂ (13 mL, Aldrich) was treated withwater (1 mL) and 30% H₂O₂ (1.1 mL, Aldrich). The mixture was stirred for30 minutes and then partitioned between 25 mL CH₂Cl₂ and 25 mL saturatedNaHCO₃ solution. The aqueous layer was extracted with 25 mL CH₂Cl₂ andthe combined CH₂Cl₂ solution washed with 1 M HCl (2×25 mL) and brine (50mL). The solution was dried (MgSO₄), filtered and evaporated to leave anorange oil. Flash chromatography (6×4 cm, 10% ether/pentane) gave enone(6) (572 mg, 2.4 mmol, 59%).

(3-Chloro-benzo[b]thiophen-2-yl)-methanol (12, FIG. 3). To an ice coldsolution of 10.0 g (47.0 mmol) of3-chloro-benzo[b]thiophene-2-carboxylic acid (11) in 200 mL of THF wasadded 47 mL of LiAlH₄ (47 mmol, 1 M/THF). After 3 hours, the reactionwas quenched by adding methanol (ca. 40 mL). The volatiles wereevaporated and the residue treated with 50 mL 1 M HCl. After stirringfor 10 minutes, the mixture was extracted with CH₂Cl₂ (3×150 mL). Thecombined CH₂Cl₂ solution was dried (MgSO₄), filtered and evaporated.Purification by flash chromatography on silica gel (10-20% ethylacetate/hexane) gave 4.32 g (21.6 mmol, 46%) of the alcohol (12).

3-Chloro-benzo[b]thiophene-2-carbaldehyde (13, FIG. 3). A solution ofalcohol 12 (4.32 g, 21.6 mmol) in 40 mL of CH₂Cl₂ was treated with 4 Amolecular sieves, NMO (3.81 g, 32.5 mmol), and TPAP (381 mg, 1.08 mmol).The reaction was stirred for 10 minutes and then dried by evaporated.Purification by flash chromatography on silica gel (2% ethylacetate/hexane) gave 3.52 g (18.3 mmol, 84%) of the aldehyde (13).

(E)-3-(3-Chloro-benzo[b]thiophen-2-yl)-acrylic acid methyl ester (14,FIG. 3). A solution of 3.52 g (18.3 mmol) of (13) in 50 mL toluene wastreated with methyl(triphenylphosphoranylidene)acetate (7.48 g, 21.9mmol). After 4 hours, saturated NaHCO₃ solution (50 mL) was added andthe mixture extracted with ethyl acetate (2×75 mL). The combined ethylacetate solution was washed with brine (50 mL), dried (Na₂SO₄), filteredand evaporated. Purification by flash chromatography on silica gel (5%ethyl acetate/hexane) provided 3.60 g (14.6 mmol, 80%) of the enoate(14).

3-(3-Chloro-benzo[b]thiophen-2-yl)-propionic acid methyl ester (15, FIG.3). A solution of 3.60 g (14.6 mmol) of (14) in 50 mL THF was treatedwith Wilkinson's catalyst (3.35 g, 3.62 mmol). The mixture was stirredunder 1 atm H₂ for 18 hours and then was filtered through celite. Thesolvent was evaporated and the residue purified by flash chromatographyon silica gel (0-2% ethyl acetate/hexane) to give 3.63 g (14.3 mmol,99%) of the saturated ester (15).

3-(3-Chloro-benzo[b]thiophen-2-yl)-propan-1-ol (16, FIG. 3). An ice coldsolution of 3.63 g (14.3 mmol) of (15) in 60 mL of ether was treatedwith LiBH₄ (621 mg, 28.5 mmol) and methanol (2 mL). After 30 minutes, 30mL of 0.5 M NaOH solution was added. The mixture was extracted withethyl acetate (2×25 mL) and the combined ethyl acetate solution waswashed with brine (50 mL), dried (MgSO₄), filtered and evaporated. Theresidue was purified by flash chromatography on silica gel (5-20% ethylacetate/hexane) to give 2.57 g (11.3 mmol, 79%) of the alcohol (16).

3-(3-Chloro-benzo[b]thiophen-2-yl)-propionaldehyde (17, FIG. 3). A −78°C. solution of oxalyl chloride (1.73 g, 13.6 mmol) in dichloromethane(20 mL) was treated with DMSO (20 mL). After 5 minutes, a solution ofalcohol (16) (2.57 g, 11.3 mmol) in dichloromethane (20 mL) was added.After another 15 minutes, triethylamine (7.1 mL, 50.6 mmol) was added.The reaction was stirred at −78° C. for 5 minutes, and warmed to roomtemperature. After 30 minutes, 100 mL water was added and the mixtureextracted with dichloromethane (3×60 mL). The combined dichloromethanesolution was dried (Na₂SO₄), filtered and evaporated. Purification byflash chromatography on silica gel (10% ethyl acetate/hexane) gave 2.11g (9.4 mmol, 83%) of the aldehyde (17).

5-(3-Chloro-benzo[b]thiophen-2-yl)-pent-1-yn-3-ol (18, FIG. 3). Asolution of aldehyde (17) (2.11 g, 9.4 mmol) in 15 mL THF was added to asolution of ethynylmagnesium bromide (28.2 mL, 14.1 mmol, 0.5 M THF) at0° C. After 1.5 hours, saturated NH₄Cl solution (75 mL) was added andthe mixture was extracted with ethyl acetate (3×50 mL). The combinedethyl acetate solution was washed with brine (50 mL) and then dried(Na₂SO₄), filtered and evaporated. Purification by flash chromatography(5-20% ethyl acetate/hexane) gave 2.20 g (8.78 mmol, 93%) of the alcohol(18).

tert-Butyl-{1-[2-(3-chloro-benzo[b]thiophen-2-yl)-ethyl]-prop-2-ynyloxy}-dimethyl-silane(19, FIG. 3). A solution of alcohol (18) (2.20 g, 8.78 mmol) indichloromethane (15 mL) was treated with DMAP (215 mg, 1.8 mmol), TBSCl(1.59 g, 10.5 mmol), and triethylamine (1.8 mL, 13.2 mmol). The reactionwas stirred for 24 hours and then saturated sodium bicarbonate solution(50 mL) was added. The mixture was extracted with dichloromethane (2×50mL) and the combined dichloromethane solution dried (Na₂SO₄), filteredand evaporated. Purification by flash chromatography (4% ethylacetate/hexane) gave 3.06 g (6.4 mmol, 73%) of the protected alcohol(19).

tert-Butyl-hex-5-ynyloxy-dimethyl-silane (26);7-(tert-Butyl-dimethyl-silanyloxy)-hept-2-yn-1-ol (27); and Acetic acid7-(tert-butyl-dimethyl-silanyloxy)-hept-2-ynyl ester (28, FIG. 4). Asolution of 7-(tert-Butyl-dimethyl-silanyloxy)-hept-2-yn-1-ol (27)(4.507 g, 21 mmol) in pyridine (20 mL) was treated with acetic anhydride(3.0 mL, 31.8 mmol). After 18 hours, the solvent was evaporated and theresidue co-evaporated with toluene. The residue was used directly in thenext step.

7-Acetoxy-hept-5-ynoic acid (29, FIG. 4). A solution of crude (28) inacetone (100 mL) was treated with Jones Reagent (18.0 mL, 41.4 mmol, 2.3M) and cooled with an ice bath. After 1 hour at room temperature, 10 mLisopropyl alcohol was added and the mixture stirred for 15 minutes. Themixture still had a brown color so another 10 mL isopropyl alcohol wasadded. After another 15 minutes, the color had not changed so themixture was filtered through celite and the filtrate evaporated invacuo. The residue was partitioned between 100 mL ether and 100 mLsaturated ammonium chloride solution. The aqueous layer was extractedwith 100 mL ether and the combined ether solution washed with brine andthen dried (MgSO₄), filtered and evaporated to leave a yellow oil (6.333g) that was used directly in the next step.

7-Hydroxy-hept-5-ynoic acid methyl ester (30, FIG. 4). The crude acid(29) (6.333 g) was treated with a 1% solution of acetyl chloride inmethanol (60 mL). After 16 hours, sodium bicarbonate (1.966 g, 23.4mmol) was added. The mixture was dried (MgSO₄), filtered through celiteand evaporated in vacuo. Purification by flash chromatography on silicagel (30-40% ethyl acetate/hexanes) gave 7-Hydroxy-hept-5-ynoic acidmethyl ester (30) (3.022 g, 19.3 mmol, 92% from7-(tert-Butyl-dimethyl-silanyloxy)-hept-2-yn-1-ol (27).

7-Iodo-hept-5-ynoic acid methyl ester (31, FIG. 4). A solution of (30)(1.347 g, 8.6 mmol) in 5 mL dichloromethane was added to a mixture oftriphenylphosphine (2.725 g, 10.4 mmol), imidazole (726 mg, 10.7 mmol),and iodine (2.602 g, 10.3 mmol) in 34 mL dichloromethane, rinsing with 5mL dichloromethane. After 40 minutes, the dichloromethane was evaporatedin vacuo to about 2 mL and the resulting mixture filtered through basicalumina, washing with 10% ethyl acetate/hexanes. Purification by flashchromatography on silica gel (10% ethyl acetate/hexanes) gave 1.878 g(7.1 mmol, 83%) of the propargyl iodide.

tert-Butyl-{(E)-1-[2-(3-chloro-benzo[b]thiophen-2-yl)-ethyl]-3-iodo-allyloxy}-dimethyl-silane(32, FIG. 5). A solution of alkyne (19) (5.547 g, 15.2 mmol) indichloromethane (50 mL) was treated with Cp₂ZrHCl (5.794 g, 22.5 mmol).The reaction was stirred for 45 minutes and then N-iodosuccinimide(4.966 g, 22.1 mmol) was added. After 15 minutes, saturated sodiumbicarbonate solution (200 mL) was added and the mixture extracted withdichloromethane (2×100 mL). The combined dichloromethane solution wasdried (MgSO₄), filtered and evaporated. Purification by flashchromatography on silica gel (0-5% ethyl acetate/hexanes) gave 6.608 g(13.1 mmol, 86%) of the vinyl iodide (32).

7-{(1R,4S,5R)-4-(tert-Butyl-dimethyl-silanyloxy)-5-[(E)-3-(tert-butyl-dimethyl-silanyloxy)-5-(3-chloro-benzo[b]thiophen-2-yl)-pent-1-enyl]-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-ynoicacid methyl ester (33, FIG. 5). A −78° C. solution of iodide (32) (675mg, 1.34 mmol) in THF (2.0 mL) was treated with tert-butyllithium (1.73mL, 2.94 mL, 1.7 M/pentane). The dark red mixture was stirred for 25minutes and then dimethylzinc (0.80 mL, 1.6 mmol, 2 M/toluene) wasadded. The solution was stirred at 0° C. for 15 minutes and thenrecooled to −78° C. A solution of enone (6) (208 mg, 0.87 mmol) in THF(1.0 mL) was added over 2 hours by syringe pump, rinsing with 0.5 mLTHF. After 30 minutes, HMPA (1.34 mL, distilled from CaH₂) was addedfollowed by a solution of propargyl iodide (31) (1.286 g, 4.83 mmol) inTHF (1.0 mL). The solution was stirred in a −40° C. bath overnight andthen 20 mL saturated ammonium chloride solution and 10 mL water wereadded. The mixture was extracted with dichloromethane (20 mL) and ethylacetate (2×20 mL). The combined organic extracts were dried (MgSO₄),filtered and evaporated. Purification by flash chromatography on silicagel (5-10% ethyl acetate/hexanes) gave 198 mg (0.27 mmol, 31%) of (33).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-ynoicacid methyl ester (34, 35, FIG. 5). A solution of (33) (198 mg, 0.27mmol) in CH₃CN (6.5 mL) was treated with HF-pyridine (1.2 mL). Thesolution was stirred for 3 hours and saturated sodium bicarbonatesolution (120 mL) was added. The mixture was extracted withdichloromethane (3×50 mL) and the combined dichloromethane solutiondried (Na₂SO₄), filtered and evaporated. Purification by flashchromatography (50% ethyl acetate/hexane) followed by preparative TLC(55% ethyl acetate/hexane) gave 55 mg (0.11 mmol, 41%) of the less polardiastereomer (34) and 51 mg (0.10 mmol, 37%) of the more polardiastereomer (35).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-ynoicacid (low R_(f) diastereomer, 36, FIG. 5). A solution of (34) (9 mg,0.017 mmol) and rabbit liver esterase (1 mg) in pH 7.2 phosphate buffer(2 mL)/CH₃CN (0.1 mL) was stirred for 17 hours. The mixture was thencoevaporated with CH₃CN to remove water and the residue purified byflash chromatography on silica gel (3-7% MeOH/CH₂Cl₂) to give 8 mg(0.016 mmol, 93%) of the acid (36).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-ynoicacid (high R_(f) diastereomer, 37, FIG. 5). A solution of (35) (12 mg,0.023 mmol) and rabbit liver esterase (1 mg) in pH 7.2 phosphate buffer(2 mL)/CH₃CN (0.1 mL) was stirred for 17 hours. TLC showed the presenceof starting material, so another 2 mg of the esterase was added. Afterstirring for another 24 hours, the reaction was complete. Work up andpurification as above for (36) gave 8 mg (0.016 mmol, 69%) of the acid(37).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid methyl ester (low R_(f) diastereomer, 38, FIG. 5). Ethanol (95%,2.5 mL) was added to NiCl₂ (50 mg, 0.39 mmol) and NaBH₄ (7 mg, 0.19mmol). The resulting black mixture was stirred for 5 minutes and thenethylenediamine (41 μL, 0.61 mmol) was added. After 15 minutes, asolution of alkyne (34) (40 mg, 0.077 mmol) in 0.5 mL 95% ethanol wasadded, rinsing with 0.5 mL ethanol. The flask was purged with H₂ andallowed to stir under 1 atm H₂ for 22 hours. The mixture was thenfiltered through celite and purified by flash chromatography on silicagel (55% ethyl acetate/hexanes) to give 17 mg (0.032 mmol, 43%) of thealkene (38).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid methyl ester (high R_(f) diastereomer 39, FIG. 5). The sameprocedure as for (36) was followed to give 17 mg (0.032 mmol, 41%) of(39).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid (low R_(f) diastereomer, 40, FIG. 5). The same procedure as abovefor (36) was used to give 9 mg (0.018 mmol, 85%) of acid (40). 300 MHz¹H NMR (CDCl₃, ppm) δ 7.73 (2H, d, J=8.4 Hz) 7.45-7.30 (2H, m) 5.8-5.6(2H, m) 5.4-5.3 (2H, m) 4.3-4.1 (1H, m) 3.57 (1H, d, J=9.7 Hz) 3.1-2.9(2H, m) 2.5-1.9 (10H, m) 1.7-1.6 (2H, m) 1.09 (3H, s) 0.89 (3H, s).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid (high R_(f) diastereomer, 41, FIG. 5). The same procedure as abovefor the (36) was used to give 9 mg (0.018 mmol, 85%) of acid (41). 300MHz ¹H NMR (CDCl₃, ppm) δ 7.73 (2H, d, J=8.8 Hz) 7.45-7.30 (2H, m)5.8-5.6 (2H, m) 5.45-5.30 (2H, m) 4.3-4.2 (1H, m) 3.61 (1H, d, J=9.7 Hz)3.1-3.0 (2H, m) 2.5-1.9 (10H, m) 1.7-1.6 (2H, m) 1.10 (3H, s) 0.90 (3H,s).

2-Benzo[b]thiophen-2-yl-ethanol (54b, FIG. 6). n-BuLi (100 mL, 160 mmol,1.6M/hexanes) was added to a −78° C. mixture of thianaphthene (54a)(17.31 g, 129 mmol) in THF (70 mL)/ether (30 mL). The mixture wasstirred at −78° C. for 2 hours and then a solution of ethylene oxide(42.86 g, 1.071 mmol) in THF (70 mL)/ether (30 mL) was added by cannulaover 15 minutes. The resulting mixture was stirred for 2 hours at −78°C. and then at room temperature for 15 hours. The mixture wasevaporated, 200 mL water was added, and the resulting mixture wasextracted with ethyl acetate (3×150 mL). The combined organic solutionwas washed with brine and then dried (Na₂SO₄), filtered, and evaporated.Purification by flash chromatography on silica gel (20% ethylacetate/hexanes) gave (54b) (13.61 g, 78 mmol, 60%).

Benzo[b]thiophen-2-yl-acetaldehyde (54c, FIG. 6). A 0° C. mixture of(54b) (8.019 g, 44.9 mmol) in 100 mL dichloromethane was treated withDess-Martin reagent (20 g, 47.2 mmol). The mixture was stirred at 0° C.for 10 minutes and at room temperature for 40 minutes. Saturated NaHCO₃solution (200 mL) and 0.1 M NaHSO₃ solution were added and the resultingmixture was extracted with ethyl acetate (3×300 mL). The combinedorganic solution was dried (Na₂SO₄), filtered and evaporated to give(54c) (8.77 g). The aldehyde was taken on crude for the next reaction.

1-Benzo[b]thiophen-2-yl-but-3-yn-2-ol (54d, FIG. 6). A solution of crude(54c) (8.77 g) in THF (100 mL) was added to a solution ofethynylmagnesium bromide (450 mL, 225 mmol, 0.5 M/THF) at 0° C. bycannula. The mixture was stirred for 1 hour at 0° C. and for 1 hour atroom temperature. The reaction was then quenched by addition of 200 mLsaturated NH₄Cl solution. The layers were separated and the aqueouslayer extracted with ethyl acetate (3×200 mL). The combined organicsolution was washed with brine and then dried (Na₂SO₄), filtered andevaporated. Purification by flash chromatography on silica gel (10% 20%ethyl acetate/hexanes) gave (54d) (7.67 g, 37.9 mmol, 84% from 5-2).

(1-Benzo[b]thiophen-2-ylmethyl-prop-2-ynyloxy)-tert-butyl-dimethyl-silane(54e, FIG. 6). DMAP (2.306 g, 18.9 mmol), TBSCl (11.502 g, 76.3 mmol)and triethylamine (5.25 mL, 37.7 mmol) were added to a solution of (54d)(7.67 g, 37.9 mmol) in dichloromethane (120 mL). After 17 hours, 150 mLof saturated NH4Cl solution was added and the layers were separated. Theaqueous layer was extracted with dichloromethane (3×100 mL) and thecombined organic solution was dried (Na₂SO₄), filtered and evaporated.Purification by flash chromatography on silica gel (4% ethylacetate/hexanes) gave (54e) (8.38 g, 26.5 mmol, 70%).

((E)-1-Benzo[b]thiophen-2-ylmethyl-3-iodo-allyloxy)-tert-butyl-dimethyl-silane(54f, FIG. 6). Cp₂ZrHCl (1.719 g, 6.67 mmol) was added to a solution of(54e) (1.372 g, 4.34 mmol) in dichloromethane (30 mL). The reaction wasstirred for 30 minutes at room temperature and N-iodosuccinimide (1.997g, 8.88 mmol) was added. After 1 hour, the reaction was poured into 100mL of saturated NaHCO₃ solution. The resulting mixture was extractedwith dichloromethane (3×75 mL) and the combined organic extracts dried(Na₂SO₄), filtered and evaporated. Purification by flash chromatographyon silica gel (2% ethyl acetate/hexanes) gave (54f) (1.7484 g, 91%).

(Z)-7-[(1R,4S,5R)-5-((E)-4-Benzo[b]thiophen-2-yl-3-hydroxy-but-1-enyl)-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl]-hept-5-enoicacid (54, 55, FIG. 6). A solution of (54f) was treated in a mannersimilar to the scheme outlined in FIG. 5 to produce (54) and (55).

(Z)-7-[(1R,4S,5R)-5-((E)-4-Benzo[b]thiophen-2-yl-3-hydroxy-but-1-enyl)-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl]-hept-5-enoicacid (2-hydroxy-ethyl)-amide (60-63, 65, FIG. 7). A solution of acid(55) (7 mg, 0.015 mmol) in DMF (0.5 mL) was treated withN-hydroxysuccinimide (6.9 mg, 0.056 mmol). The mixture was stirred for 5minutes and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDCl, 20.7 mg, 0.11 mmol) was added. After stirring for 7hours, 2-aminoethanol (5 μL, 0.083 mmol) was added and the mixturestirred further for 16 hours. Ethyl acetate (50 mL) was added and themixture washed with water (3×50 mL) and brine (50 mL). The organic layerwas dried (Na₂SO₄), filtered and evaporated. Purification by flashchromatography on silica gel (5% methanol/dichloromethane) followed bypreparative thin layer chromatography (10% methanol/dichloromethane)gave amide (65) (5 mg, 0.010 mmol, 65%). Amides (60-63) were prepared ina similar manner.

(Z)-7-[(1R,4S,5R)-5-((E)-4-Benzo[b]thiophen-2-yl-3-hydroxy-but-1-enyl)-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl]-hept-5-enoicacid amide (69, FIG. 7). A solution of acid (55) (9 mg, 0.02 mmol) indichloromethane (0.2 mL) was treated with triethylamine (15 μL, 0.11mmol). The solution was cooled to 0° C. and after 10 minutes, ethylchloroformate (7 μL, 0.073 mmol) was added. The solution was stirredfurther for 1 hour at 0° C. and then concentrated aqueous ammoniumhydroxide solution was added (10 μL, 0.26 mmol). The reaction wasstirred at room temperature overnight and then quenched by addition of0.5 M HCl (7 mL). The mixture was extracted with ethyl acetate (3×30mL), the combined ethyl acetate solution washed with saturated NaHCO₃solution (20 mL) and brine (20 mL), and then dried (Na₂SO₄), filteredand evaporated. Purification by flash chromatography on silica gel(2%-6% methanol/dichloromethane) gave the title amide (2.6 mg, 28%).

Example 2 Compounds of Formula I Promote Synthesis of TGF-β and VEGF

This example illustrates that compounds of formula I promote formationof blood vessels.

To test whether the compounds of Formula I promote the formation ofblood vessels, and incisional wound model was employed over a 14-dayperiod. Sprague-Dawley rats (180-200 g) were anesthetized, the backsshaved, and a 2-cm long incision was made on the back skin of rats understerile conditions, reaching the deep fascia. The wound was immediatelyclosed with 4-0 sutures. The animals were topically treated with vehicleor Compound 1 at 0.004% twice daily. The vehicle contains ethanol 30%,propylene glycol 12%, dipropylene glycol 5%, benzyl alcohol 5%, glycerol3% and normal saline 45%. Biopsy samples of skin wound tissues wereanalyzed with Western blots day-7 and day-14 post-surgery. Tissuesamples about 1 mm thick were taken from both sides of the wound.Protein was extracted, and FGF-2/VEGF expression was monitored, withWestern blot analysis.

Compound 1 treatment significantly enhanced the expression of FGF-2 by60%, as normalized with β-actin (a house-keeping gene) by day-7 of theexperiment (P=0.004). This significantly different increase in FGF-2synthesis persisted for the whole study period (P=0.04). VEGF expressionincreased 25% as normalized with β-actin, by Compound 1 treatment onday-7 (P=0.039). This significantly different increase in synthesisreturned to the baseline by day-14. Such an increase in FGF-2 and VEGFis consistent with their role in new blood vessel formation. Theup-regulation of FGF-2 and VEGF by Compound 1 treatment was alsoobserved in studies using an oral ulcer wound model.

Example 3 Adipose Tissue Transplant for Breast Defect Correction

This example illustrates the use of compositions and methods disclosedherein for a breast defect correction.

A 32-year-old woman presented with complaints that the medial portionsof her breast implants were visible, which accentuated the “bony”appearance of her sternum. In addition she felt her breast were too farapart. Pre-operative evaluation of the person includes routine historyand physical examination in addition to thorough informed consentdisclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor a soft tissue replacement method using the compositions and methodsdisclosed herein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the lateral and medial thigh regions.The harvested area is injected subcutaneously with a standard tumescentfluid solution containing a saline solution, 0.5% lidocaine, and about0.001% epinephrine. Using an 11-blade scalpel, a small puncture wound ismade in order to transverse the dermis. The blade is turned 360 degreesto complete the wound. A two-holed blunt harvesting cannula (3 mm innerdiameter) connected to a vacuum pump at low negative pressure (0.5 atm)is then inserted into the subcutaneous adipose tissue plane. The cannulais then moved throughout the plane to disrupt the connective tissuearchitecture. The volume of aspirate obtained is about 300 mL. Theharvest adipose tissue is processed by centrifugation at 3,000 g for 3minutes to separate healthy adipocytes and regenerative cells fromblood, infiltration fluid and cell debris.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with an effective amount ofHA and the processed adipose tissue. The amount of compound added is anamount sufficient to promote formation of a blood supply sufficient tosupport the transplanted tissue. This composition is then transferred to3 mL syringes. One-holed blunt infiltration cannulas (3 mm innerdiameter) are used to place the adipose tissue subcutaneously over thelateral sternum and medial breast bilaterally, 70 mL on the right and 50mL on the left. The adipose tissue is administered in a tear likefashion to increase the surface area to volume ratio.

Alternatively, the adipose tissue is first administered into theindividual, and a composition comprising compound of formula I, such as,e.g., Compound 1, Compound 2, Compound 3, Compound 4, and/or Compound 5and HA is subsequently administered into the same, or in the vicinityof, the region where the adipose tissue was implanted. Alternatively,the adipose tissue is first administered into the individual, and acompound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5 and a composition comprising HA aresubsequently administered (either at the same time, or sequentiallyi.e., compound of formula I first and then HA or HA first and thencompound of formula I) into the same, or in the vicinity of, the regionwhere the adipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that her quality of life has improved.

Example 4 Adipose Tissue Transplant for Breast Augmentation

This example illustrates the use of compositions and methods disclosedherein for a breast augmentation.

A 28-year-old woman presented micromastia or breast hypoplasia.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for a softtissue replacement method using the compositions and methods disclosedherein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the lateral and medial thigh regions.Using a 10-blade scalpel, a small puncture wound is made in order totransverse the dermis. The blade is turned 360 degrees to complete thewound. A two-holed blunt harvesting cannula (3 mm inner diameter)connected to a syringe is then inserted into the subcutaneous adiposetissue plane. The cannula is then moved throughout the plane to disruptthe connective tissue architecture. The volume of aspirate obtained isabout 600 mL. The harvest adipose tissue is processed by centrifugationat 2,700 g for 5 minutes to separate healthy adipocytes and regenerativecells from blood, infiltration fluid and cell debris. The centrifugedadipose tissue is then washed once is a Ringer's saline solution withlactone.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with an effective amount ofHA and the processed adipose tissue. The amount of compound added is anamount sufficient to promote formation of a blood supply sufficient tosupport the transplanted tissue. This composition is then transferred to10 mL syringes. One-holed blunt infiltration cannulas (3 mm innerdiameter) are used to place the adipose tissue subcutaneously usingaxillary, periareolar, and inframammary routes bilaterally, 190 mL onthe right and 245 mL on the left. The adipose tissue is administered ina tear like fashion to increase the surface area to volume ratio.

Alternatively, the adipose tissue is first administered into theindividual, and then a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5 and a compositioncomprising HA are subsequently administered (either at the same time, orsequentially i.e., compound of formula I first and then HA or HA firstand then compound of formula I) into the same, or in the vicinity of,the region where the adipose tissue was implanted. As anotheralternative, a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5 is firstadministered into the individual, and then adiposes tissue and acomposition comprising HA are subsequently administered (either at thesame time, or sequentially i.e., adipose tissue first and then HA or HAfirst and then adipose tissue) into the same, or in the vicinity of, theregion where the compound was administered. As yet another alternative,composition comprising HA is first administered into the individual, andthen adiposes tissue and a compound of formula I, such as, e.g.,Compound 1, Compound 2, Compound 3, Compound 4, and/or Compound 5 aresubsequently administered (either at the same time, or sequentiallyi.e., adipose tissue first and then compound or compound first and thenadipose tissue) into the same, or in the vicinity of, the region wherethe HA was administered.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that her quality of life has improved.

Example 5 Adipose Tissue Transplant for Breast Disorder

This example illustrates the use of compositions and methods disclosedherein for a breast disorder.

A 29-year-old woman presented with bilaterial tiberous breast deformity.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for a softtissue replacement method using the compositions and methods disclosedherein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the abdomen, buttock, lateral andmedial thigh, and trochanter regions. Using a 12-blade scalpel, a smallpuncture wound is made in order to transverse the dermis. The blade isturned 360 degrees to complete the wound. A two-holed blunt harvestingcannula (3 mm inner diameter) connected to a syringe is then insertedinto the subcutaneous adipose tissue plane. The cannula is then movedthroughout the plane to disrupt the connective tissue architecture. Thevolume of aspirate obtained is about 1,400 mL.

The harvested adipose tissue is divided into two, approximately equalportions. One portion is processed by gravity sedimentation to separatehealthy adipocytes and regenerative cells from blood, infiltration fluidand cell debris. The other portion is used to isolate regenerativecells. This portion is digested with 0.075% collagenase in bufferedsaline for 30 minutes on a shaker at 37° C. Regenerative cells are thenseparated from mature adipocytes and connective tissue by centrifugingat 800 g for 10 minutes. The pellet containing the regenerative cells isthen washed three times with buffered saline. The washed regenerativecells are then added back to the sediment purified adipose tissue.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with an effective amount ofHA and the processed adipose tissue. The amount of compound added is anamount sufficient to promote formation of a blood supply sufficient tosupport the transplanted tissue. This composition is then transferred to10 mL syringes. One-holed blunt infiltration cannulas (3 mm innerdiameter) are used to place the adipose tissue subcutaneously inmultiple planes axillary, periareolar, and inframammary routesbilaterally, 380 mL on the right and 370 mL on the left. The adiposetissue is administered in a tear like fashion to increase the surfacearea to volume ratio.

Alternatively, the adipose tissue is first administered into theindividual, and a composition comprising compound of formula I, such as,e.g., Compound 1, Compound 2, Compound 3, Compound 4, and/or Compound 5and HA is subsequently administered into the same, or in the vicinityof, the region where the adipose tissue was implanted. Alternatively,the adipose tissue is first administered into the individual, and acompound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5 and a composition comprising HA aresubsequently administered (either at the same time, or sequentiallyi.e., compound of formula I first and then HA or HA first and thencompound of formula I) into the same, or in the vicinity of, the regionwhere the adipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that her quality of life has improved.

Example 6 Adipose Tissue Transplant for Facial Defects of Cheek

This example illustrates the use of compositions and methods disclosedherein for a facial disorder.

A 28-year-old woman presented with a lean face. She felt her face lookedold, sad and bitter because of the less fullness of her cheek contour.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for a softtissue replacement method using the compositions and methods disclosedherein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the abdominal region. The harvestedarea is injected subcutaneously with a standard tumescent fluid solutioncontaining a saline solution, 0.08% lidocaine, and about 0.001%epinephrine. Using an 11-blade scalpel, a small puncture wound is madein order to transverse the dermis. The blade is turned 360 degrees tocomplete the wound. A two-holed blunt harvesting cannula (2.5 mm innerdiameter) connected to a 60 mL syringe is then inserted into thesubcutaneous adipose tissue plane. The cannula is then moved throughoutthe plane to disrupt the connective tissue architecture. The volume ofaspirate obtained is about 50 mL. The harvest adipose tissue isprocessed by washing the harvested tissue with saline to removelidocaine, oil and residual blood. The washed tissue is thencentrifugation at 100 g for 2 minutes to separate healthy adipocytes andregenerative cells from any remaining blood, infiltration fluid and celldebris.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with an effective amount ofHA and the processed adipose tissue. The amount of compound added is anamount sufficient to promote formation of a blood supply sufficient tosupport the transplanted tissue. This composition is then transferred to20 mL syringes. One-holed blunt infiltration cannulas (3 mm innerdiameter) are used to place about 15 mL of adipose tissue subcutaneouslyand under superficial musculoaponeurotix system into the left and rightcheeks.

Alternatively, the adipose tissue is first administered into theindividual, and a composition comprising compound of formula I, such as,e.g., Compound 1, Compound 2, Compound 3, Compound 4, and/or Compound 5and HA is subsequently administered into the same, or in the vicinityof, the region where the adipose tissue was implanted. Alternatively,the adipose tissue is first administered into the individual, and acompound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5 and a composition comprising HA aresubsequently administered (either at the same time, or sequentiallyi.e., compound of formula I first and then HA or HA first and thencompound of formula I) into the same, or in the vicinity of, the regionwhere the adipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure because she looked younger. Approximately onemonth after the procedure, the woman indicates that her quality of lifehas improved.

Example 7 Adipose Tissue Transplant for Facial Defects of Eyelids

This example illustrates the use of compositions and methods disclosedherein for a facial disorder.

A 37-year-old woman presented with sunken eyes and this appearance madeher look old and fierce. Pre-operative evaluation of the person includesroutine history and physical examination in addition to thoroughinformed consent disclosing all relevant risks and benefits of theprocedure. The physician evaluating the individual determines that sheis a candidate for a soft tissue replacement method using thecompositions and methods disclosed herein.

To adipose tissue is harvested from the woman as described in Example 6.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with an effective amount ofHA and the processed adipose tissue. The amount of compound added is anamount sufficient to promote formation of a blood supply sufficient tosupport the transplanted tissue. This composition is then transferred to20 mL syringes. One-holed blunt infiltration cannulas (3 mm innerdiameter) are used to place about 2.5 mL of adipose tissuesubcutaneously and under superficial musculoaponeurotix system into theupper eyelid regions.

Alternatively, the adipose tissue is first administered into theindividual, and a composition comprising compound of formula I, such as,e.g., Compound 1, Compound 2, Compound 3, Compound 4, and/or Compound 5and HA is subsequently administered into the same, or in the vicinityof, the region where the adipose tissue was implanted. Alternatively,the adipose tissue is first administered into the individual, and acompound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5 and a composition comprising HA aresubsequently administered (either at the same time, or sequentiallyi.e., compound of formula I first and then HA or HA first and thencompound of formula I) into the same, or in the vicinity of, the regionwhere the adipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure because she looked younger. Approximately onemonth after the procedure, the woman indicates that her quality of lifehas improved.

Example 8 Adipose Tissue Transplant for Facial Defects

This example illustrates the use of compositions and methods disclosedherein for a breast disorder.

A 33-year-old woman presented with depressed bilaterial temporal andcheek areas. Pre-operative evaluation of the person includes routinehistory and physical examination in addition to thorough informedconsent disclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor a soft tissue replacement method using the compositions and methodsdisclosed herein.

To adipose tissue is harvested from the woman as described in Example 6,except that 200 mL of adipose tissue is collected.

The harvested adipose tissue is divided into two, approximately equalportions. One portion is processed by saline washing and centrifugationas described in Example 6. The other portion is used to isolateregenerative cells. This portion is digested with 0.075% collagenase inbuffered saline for 30 minutes on a shaker at 37° C. Regenerative cellsare then separated from mature adipocytes and connective tissue bycentrifuging at 800 g for 10 minutes. The pellet containing theregenerative cells is then washed three times with buffered saline. Thewashed regenerative cells are then added back to the purified adiposetissue.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with an effective amount ofHA and the processed adipose tissue. The amount of compound added is anamount sufficient to promote formation of a blood supply sufficient tosupport the transplanted tissue. This composition is then transferred to10 mL syringes. One-holed blunt infiltration cannulas (3 mm innerdiameter) are used to place about 16 mL of adipose tissue subcutaneouslyand under superficial musculoaponeurotix system into the left and righttemporal and cheeks regions.

Alternatively, the adipose tissue is first administered into theindividual, and a composition comprising compound of formula I, such as,e.g., Compound 1, Compound 2, Compound 3, Compound 4, and/or Compound 5and HA is subsequently administered into the same, or in the vicinityof, the region where the adipose tissue was implanted. Alternatively,the adipose tissue is first administered into the individual, and acompound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5 and a composition comprising HA aresubsequently administered (either at the same time, or sequentiallyi.e., compound of formula I first and then HA or HA first and thencompound of formula I) into the same, or in the vicinity of, the regionwhere the adipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure because she looked younger. Approximately onemonth after the procedure, the woman indicates that her quality of lifehas improved.

Example 9 Adipose Tissue Transplant to Treat Stress Urinary Incontinence

This example illustrates the use of compositions and methods disclosedherein for treating stress urinary incontinence.

A 55 year old man presents with urinary incontinence. Pre-operativeevaluation of the patient includes routine history and physicalexamination in addition to thorough informed consent disclosing allrelevant risks and benefits of the procedure. The physician evaluatingthe individual determines that he is a candidate for a soft tissuereplacement method using the compositions and methods disclosed herein.

To begin the procedure, adipose tissue is harvested from the man. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the abdomen, and lateral and medialthigh regions. Using a 12-blade scalpel, a small puncture wound is madein order to transverse the dermis. The blade is turned 360 degrees tocomplete the wound. A two-holed blunt harvesting cannula (3 mm innerdiameter) connected to a syringe is then inserted into the subcutaneousadipose tissue plane. The cannula is then moved throughout the plane todisrupt the connective tissue architecture. The volume of aspirateobtained is about 900 mL.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with an effective amount ofHA and the processed adipose tissue. The amount of compound added is anamount sufficient to promote formation of a blood supply sufficient tosupport the transplanted tissue. This composition is then transferred to20 mL syringes. One-holed blunt infiltration cannulas (14-gauge) areused to place about 800 mL of adipose tissue transdermally into thebladder neck and proximal urethra regions.

Alternatively, the adipose tissue is first administered into theindividual, and a composition comprising compound of formula I, such as,e.g., Compound 1, Compound 2, Compound 3, Compound 4, and/or Compound 5and HA is subsequently administered into the same, or in the vicinityof, the region where the adipose tissue was implanted. Alternatively,the adipose tissue is first administered into the individual, and acompound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5 and a composition comprising HA aresubsequently administered (either at the same time, or sequentiallyi.e., compound of formula I first and then HA or HA first and thencompound of formula I) into the same, or in the vicinity of, the regionwhere the adipose tissue was implanted.

The individual is monitored after the procedure. Approximately threedays after the transplant, the man indicates that he experiences adecreased frequency of incontinence. Approximately one month after theprocedure, the individual indicates that his quality of life hasimproved. The physician evaluates the engrafted tissue and determinesthat the long-term engraftment was successful.

Example 10 Adipose Tissue Transplant for Breast Defect Correction

This example illustrates the use of compositions and methods disclosedherein for a breast defect correction.

A 56-year-old woman presents with a surgically removed breast due tocancer. Pre-operative evaluation of the person includes routine historyand physical examination in addition to thorough informed consentdisclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor a soft tissue replacement method using the compositions and methodsdisclosed herein.

To begin the procedure, a breast mound is formed using a TRAM-flapprocedure. In this procedure, a portion of abdomen tissue, includingskin, adipose tissue, minor muscles and connective tissues, is takenfrom the patient's abdomen and transplanted onto the breast site. Thistissue is then used to create a breast mound.

A composition comprising a compound of formula I, such as, e.g.,Compound 1, Compound 2, Compound 3, Compound 4, and/or Compound 5 and aneffective amount of HA is then administered into the breast moundregion. The amount of compound administered is an amount sufficient topromote formation of a blood supply sufficient to support thetransplanted tissue.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that her quality of life has improved. Subsequentsurgery is performed to create a nipple and areola.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of the present invention are described herein,including the best mode known to the inventors for carrying out theinvention. Of course, variations on these described embodiments willbecome apparent to those of ordinary skill in the art upon reading theforegoing description. The inventor expects skilled artisans to employsuch variations as appropriate, and the inventors intend for the presentinvention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalentsof the subject matter recited in the claims appended hereto as permittedby applicable law. Moreover, any combination of the above-describedembodiments in all possible variations thereof is encompassed by theinvention unless otherwise indicated herein or otherwise clearlycontradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentinvention are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe invention are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the present invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. All methods described herein can be performed in any suitableorder unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein is intended merely to betterilluminate the present invention and does not pose a limitation on thescope of the invention otherwise claimed. No language in the presentspecification should be construed as indicating any non-claimed elementessential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

What is claimed is:
 1. A composition comprising: a) an adipose tissue;b) a hydrogel material comprising a glycosaminoglycan polymer; c) acompound having the structure of compound 24,

a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof.
 2. The composition of claim 1, whereinthe compound is incorporated into a drug delivery platform comprising abiodegradable polymer matrix.
 3. The composition of claim 1, wherein thehydrogel material is a porous material or solid particles.
 4. Thecomposition of claim 1, wherein glycosaminoglycan polymer is ahyaluronan polymer.
 5. A method of treating a soft tissue condition ofan individual, the method comprising the step of administering to a siteof the soft tissue condition a composition comprising adipose tissue, ahydrogel material comprising a glycosaminoglycan polymer, and acompound, the compound having the structure of compound 24,

a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof; wherein administration of the compositionpromotes formation of a blood supply sufficient to support thetransplanted tissue, thereby treating the soft tissue site.
 6. Themethod of claim 5, wherein the soft tissue condition is a breast tissuecondition, a facial tissue condition, a neck condition, a skincondition, an upper arm condition, a lower arm condition, a handcondition, a shoulder condition, a back condition, a torso includingabdominal condition, a buttock condition, an upper leg condition, alower leg condition including calf condition, a foot condition includingplantar fat pad condition, an eye condition, or a genital condition. 7.The method of claim 6, wherein the breast tissue condition is a breastimperfection, a breast defect, a breast augmentation, or a breastreconstruction.
 8. The method of claim 6, wherein the facial tissuecondition is a facial imperfection, a facial defect, a facialaugmentation, or a facial reconstruction.
 9. The method of claim 6,wherein the facial soft tissue condition is a dermal divot, a sunkencheek, a thin lip, a nasal imperfection or defect, a retro-orbitalimperfection or defect, a facial fold, a facial line, a facial wrinkle,or other size, shape or contour imperfection or defect of the face. 10.The method of claim 5, wherein the soft tissue condition is urinaryincontinence, fecal incontinence, or gastroesophageal reflux disease(GERD).
 11. A method of treating a soft tissue condition of anindividual, the method comprising the steps of a) administering adiposetissue to a site of the soft tissue condition; b) administering aneffective amount of a hydrogel material comprising a glycosaminoglycanpolymer to the site of the soft tissue condition; and c) administering acompound to the site of the soft tissue condition, the compound havingthe structure of compound 24,

a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof; wherein administration of the compoundpromotes formation of a blood supply sufficient to support thetransplanted tissue, thereby treating the soft tissue site.
 12. Themethod of claim 11, wherein the glycosaminoglycan polymer is ahyaluronan polymer.
 13. The method of claim 11, wherein the hydrogelmaterial is administered at the same time as, before, or after thecompound is administered.
 14. The method of claim 11, wherein the softtissue condition is a breast tissue condition, a facial tissuecondition, a neck condition, a skin condition, an upper arm condition, alower arm condition, a hand condition, a shoulder condition, a backcondition, a torso including abdominal condition, a buttock condition,an upper leg condition, a lower leg condition including calf condition,a foot condition including plantar fat pad condition, an eye condition,or a genital condition.
 15. The method of claim 14, wherein the breasttissue condition is a breast imperfection, a breast defect, a breastaugmentation, or a breast reconstruction.
 16. The method of claim 14,wherein the facial tissue condition is a facial imperfection, a facialdefect, a facial augmentation, or a facial reconstruction.
 17. Themethod of claim 14, wherein the facial soft tissue condition is a dermaldivot, a sunken cheek, a thin lip, a nasal imperfection or defect, aretro-orbital imperfection or defect, a facial fold, a facial line, afacial wrinkle, or other size, shape or contour imperfection or defectof the face.
 18. The method of claim 11, wherein the soft tissuecondition is urinary incontinence, fecal incontinence, orgastroesophageal reflux disease (GERD).